RUNX1 mutations being one of the most common. Tyrosine kinase inhibitor therapy has only limited efficacy in BP-CML, and characterization of more defined molecular subtypes is warranted in order to design better treatment modalities for this poor prognosis patient group. Using whole-exome and RNA sequencing we demonstrate that PHF6 and BCORL1 mutations, IKZF1 deletions, and AID/RAG-mediated rearrangements are enriched in RUNX1mut BP-CML leading to typical mutational signature. On transcriptional level interferon and TNF signaling were deregulated in primary RUNX1mut CML cells and stem cell and B-lymphoid factors upregulated giving a rise to distinct phenotype. This was accompanied with the sensitivity of RUNX1mut blasts to CD19-CAR T cells in ex vivo assays. High-throughput drug sensitivity and resistance testing revealed leukemia cells from RUNX1mut patients to be highly responsive for mTOR-, BCL2-, and VEGFR inhibitors and glucocorticoids. These findings were further investigated and confirmed in CRISPR/Cas9-edited homozygous RUNX1−/− and heterozygous RUNX1−/mut BCR-ABL positive cell lines. Overall, our study provides insights into the pathogenic role of RUNX1 mutations and highlights personalized targeted therapy and CAR T-cell immunotherapy as potentially promising strategies for treating RUNX1mut BP-CML patients.Blast-phase chronic myeloid leukemia (BP-CML) is associated with additional chromosomal aberrations,  RUNX1, also known as core binding factor subunit alpha (CBFA2), is a transcription factor (TF) and an essential component of the core binding factor complex that plays a key role in hematopoiesis [RUNX1 gene are commonly encountered in a variety of hematological malignancies [RUNX1 germline mutations are associated with familial platelet disorders (FPD) with predisposition to hematological malignancies [RUNX1 is affected by a range of chromosomal rearrangements resulting in fusions with multiple partners [RUNX1-RUNX1T1 translocation in 15% of AML patients [ETV6-RUNX1 translocation in 25% of BCP-ALL patients [RUNX1-MECOM in therapy-related MDS/AML patients [ETV6-RUNX1 ALL, it has been reported that the aberrant RAG recombination activity mediates off-target deletions and is the driver mutagenic mechanism [lpha CBFA, is a trgnancies . In acutpartners . These ipatients . In the echanism . In normechanism , 10.RUNX1 mutations are frequent among hematological malignancies like AML [RUNX1 (RUNX1mut AML) is a provisional entity which accounts for 10% of the newly diagnosed patients and associates with an inferior prognosis [RUNX1 mutations are frequently encountered in AML patients with minimal differentiation (AML-M0), where it demonstrates a unique molecular signature with upregulation of B-lymphoid genes [-AML [RUNX1mut AML shows associations with mutations affecting spliceosome (SRSF2 and SF3B1), epigenetic modifiers (ASXL1 and EZH2), and PHF6 and BCOR genes [FLT3-ITD and MLL-PTD frequently coexist with RUNX1 mutations, while fusion genes and NPM1 mutations are mutually exclusive with RUNX1 mutations [Somatic like AML , ALL [12like AML , and MDSlike AML . AML witrognosis , 15. RUNid genes . Aberran;21)-AML , represe;21)-AML . RUNX1muOR genes , 20. FurRUNX1 mutations have also been found in CML patients and linked to disease progression and inferior treatment responses [RUNX1 mutations were identified as recurrent events in BP-CML (3 out of 20 patients) [RUNX1 mutations can contribute to blast transformation of CML [RUNX1 mutations in BP-CML. We therefore investigated the mutational profiles of RUNX1-mutated (RUNX1mut) and wild-type (RUNX1wt) BP-CML patients with whole-exome and RNA sequencing and integrated public genomic data of BP-CML patients to increase accuracy. This approach allowed us to enlighten a novel mutagenesis role of RUNX1 mutations that is coupled with the activation of AID/RAG axis. Gene expression profiling demonstrated characteristic transcriptional programming in RUNX1mut cases including upregulation of stem cell and B-lymphoid genes. Using drug sensitivity profiling of primary leukemia cells and CRISPR/Cas9 RUNX1 gene-edited CML cell lines, we identified novel effective targeted therapies and CD19-CAR T cells as a promising immunotherapeutic option. Our data provide a comprehensive genomic and functional characterization of RUNX1mut BP-CML.esponses –24. In oatients) . In concn of CML , 27. NevClinical and hematological features of BP-CML patients are summarized in Supplementary Table P210-BCR-ABL1-GFP were a gift from Prof. Nikolas von Bubnoff, Universitätsklinikum Freiburg, Germany. K562 was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures). Both cell lines were cultured in RPMI-1640 (Lonza) supplemented with 10% FBS, 2-mM L-glutamine (Lonza), and 100-U/mL penicillin and 100-μg/mL streptomycin (Gibco).Baf3 cells transfected with Patient samples (bone marrow mononuclear cells (BMNCs)) and cell lines were stained with relevant panels of antibodies as indicated in the Supplementary materials, using recommended manufacturer protocols for surface antibodies staining. Cells were acquired with the FACS Verse and analyzed with FlowJo software . All antibodies were purchased from BD Biosciences, San Diego, CA, USA.Genomic DNA was extracted from BP-CML patients’ BMNCs. Skin samples were collected and used as germline controls to identify somatic mutations. WES protocol has been described in the earlier study . The meaThe oncology compounds library, employed to test patient samples, consisted of 125 FDA/EMA anticancer approved drugs and 127 investigational and preclinical compounds. For cell lines, a comprehensive library of 528 compounds  was used. Drugs were tested in five increasing concentrations over a 10,000-fold range. For drug combination testing, the selected drugs were tested with dose–response matrices comprising seven different concentrations. DSRT was performed as previously described , and quaBCR-ABL1 cells were transfected with pU6-(BbsI)-CBh-Cas9-T2A-mCherry (Addgene plasmid#64324) expressing CRISPR-Cas9 and sgRNA targeting exon-4 of runx1 gene using Fugene HD transfection reagent (Promega). All of sgRNA, primers, oligos, plasmids, and antibodies are listed in Supplementary Table Baf3-CAR T cells were manufactured and the ex vivo cytotoxicity assay was performed as previously described , 31 and t test, Mann–Whitney U-test, Fisher Exact test, Spearman correlation, Pearson’ correlation tests, and Fisher’s Exact Test with simulated p value on 1e + 07 replicates were computed using GraphPad Prism 7 software or R 3.5.0.Two-tailed Student n = 1), relapse (n = 2), or both (n = 1). In our WES cohort  and one nonsense mutation (p.K117*), that were all located in the Runt domain  (Supplementary Table RUNX1mut BP-CML patients showed a notable population of plasmacytoid dendritic cells (pDCs) in contrast to RUNX1wt group  using WES  in three patients  was seen also in diagnosis (CP) sample , with acquisition of loss of heterozygosity and loss of the wild-type allele prior sampling at BP (VAF = 99%)  showed no significant differences in the mutational load or structural variants (SV) between RUNX1mut and RUNX1wt patient samples . RAG heptamer was demonstrated in 31 sites involving one or both ends of 23/32 (71.9%) of breakpoints in RUNX1mut patients  compared with 20 sites involving 16/39 (41%) of breakpoints in RUNX1wt patients  of breakpoints, E value = 8.0 × 10–46)  and lymphoid progenitors  were upregulated, whereas markers of megakaryopoiesis, erythropoiesis, and granulopoiesis  were downregulated in RUNX1mut patients , CD69, and multiple HLA-DR and TLR molecules, in addition to cytokine receptors   pathways were enriched in the downregulated sets. Expression profile of RUNX1mut BP-CML patients shared similarities with CBF-related AML in contrast with NPM1mut-AML and AML with granulocytic or megakaryocytic differentiation  showed upregulation of interferon alpha and gamma signaling, antigen processing and presentation, TNF and MAPK signaling pathways in ted Fig. , e. RUNXRUNX1mut-induced transcriptional changes can also be demonstrated in lymphoid-BP phenotype. We analyzed data of lymphoid-BP patients from Branford et al. [n = 16 patients, RUNX1mut = 7, RUNX1wt = 9). Lymphoid-BP samples showed clustering according to RUNX1 mutation status. Upregulation of several genes, including BAALC, CD133, ST18, and FLT4, was comparable to RUNX1mut myeloid-BP profiles. Furthermore, GSEA demonstrated similarities of upregulated pathways between RUNX1mut lymphoid-BP and CBF-related AML in contrast to NPM1mut-AML, highlighting RUNX1mut-specific transcriptional signature , we investigated whether d et al.   and FLT4 gene (VEGFR3 receptor)   and RUNX1wt (n = 2) patients with imatinib in combination with dexamethasone, everolimus, cobimetinib, axitinib as well as venetoclax in a dose-dependent manner to investigate potential synergistic drug activities  and a RUNX1−/mut cell line with an in-frame deletion (−3), predicted to have a deleterious effect on protein function, together with an out of frame (−1) deletion (heterozygous deletion) using CRISPR-cas9 technology, that was validated using western blot of RUNX1 protein  nor parental cell line  and BET inhibitors , which were not tested in the patient samples  with and without imatinib using flow cytometry  and one myeloid-BP patient (mut2) with aberrant CD19 expression on 25% of blasts. In a 24-h coculture experiment, CD19-CAR T cells were able to induce killing of blasts at effector–target (E–T) (CAR T cells: CD34+ cell) ratio as low as 1:8 at a variable extent (13–50%). The demonstrated cytotoxic activity of CD19-CAR T cells was specific in contrast with mock-CAR T cells   Fig. . CocultunM) Fig. .Fig. 6ExRUNX1-mutated patients (n = 2), CD19-CAR T cells-induced cytotoxicity was modest, highlighting the specificity of CAR T cells activity , and the sequencing method used [ABL1 and RUNX1 mutations were the most common mutations. In our own discovery cohort, we identified four mutations of RUNX1, that were located within the Runt domain, in line with reports of BP-CML and AML [RUNX1 protein functions, including CBFB dimerization and DNA binding, in addition to leukemia transformation [RUNX1 mutation was the sole leukemia-associated mutation identified both in CP (SNV) and progression (SNV and LOH) samples. Giustacchini et al. [RUNX1 mutation in both CP stem cells (SCs) and BP-SCs of a lymphoid-BP patient. RUNX1mut CP-SCs demonstrate transcriptional similarities with BP-SCs, rather than with RUNX1wt CP-SCs.The incidence of hod used , 24, 27. and AML , 26, 27.ormation . In a myi et al. . similarRUNX1 aberrations contribute to mutagenesis and leukemic predisposition [CETN2 and MLH1, in RUNX1mut BP-CML. We identified RAG-mediated recombination to be associated with RUNX1mut. RUNX1 is important for RAG function in early T-cell development [AID/RAG activity is implicated in lymphoid malignancies, namely in ETV6-RUNX1 ALL [AID/RAG, which can increase genetic vulnerability [RUNX1mut patients exhibited significant association with AID/RAG-related SHM signature and enrichment of RAG-RSS compared with cells from RUNX1wt patients. We demonstrated presence of RAG off-target activity in an IKZF1 deletion in RUNX1mut BP-CML, like previously reported in Philadelphia-positive ALL (Ph-ALL) [AID expression was suggested to contribute to lymphoid progression in CML [RUNX1mut myeloid-BP patient with exceptionally high RAG expression and aberrant lymphoid markers phenotype supporting the role of RUNX1mut-induced RAG activity.position , and assposition . Likewiselopment . AberranUNX1 ALL . Our datrability . Leukemi(Ph-ALL) . AID expn in CML . Recentln in CML . InteresRUNX1mut BP-CML shares several genomic features with RUNX1mut AML, underscoring similarity of RUNX1mut across leukemias. RUNX1mut BP-CML exhibited other mutations in BCORL1 and PHF6 genes, as well as IKZF1 deletions, comparable to the mutational landscape of RUNX1mut AML [RUNX1mut BP-CML was another similarity with RUNX1mut AML [RUNX1 mutations in FPD/AML [RUNX1mut BP-CML showed aberrant expression of lymphoid antigens  in myeloid-BP patients and overexpression of lymphoid TFs and markers similar to RUNX1mut AML [-AML to relate with PAX5 overexpression [PAX5 in RUNX1mut BP-CML patients, in concord with data from RUNX1mut AML [RUNX1mut BP-CML patients showed upregulation of many pDC markers. RUNX1 is a key TF in pDC development through regulation of IRF8 [RUNX1 mutations and expression of lymphoid antigens, comparable to RUNX1mut BP-CML. Noteworthy, a study including 47 RUNX1mut-AML patients showed that RUNX1mut blasts shared a common gene expression signature in contrast with RUNX1wt blasts, and transcriptional differences between missense and nonsense RUNX1 mutations were demonstrated in some RUNX1 target genes [1mut AML , 21, 43.1mut AML , 45. Dow FPD/AML . Further1mut AML . Aberranpression . We demo1mut AML . RUNX1mu of IRF8 . A recen of IRF8 , demonstet genes  will addRUNX1mut BP-CML patients, including mTOR inhibitors, glucocorticoids, VEGFR inhibitors, and BCL2 inhibitors. VEGFR and mTOR inhibitors are active in CBF-AML [RUNX1mutAML, reflecting shared RUNX1mut signature. In RUNX1mutAML, glucocorticoid sensitivity is associated with RUNX1 mutations and wild-type RUNX1 activity [RUNX1mut cell lines were recently suggested as a targeted therapy for RUNX1mut AML [RUNX1mut blasts, representing promising treatment strategies for RUNX1mut BP-CML. A strong evidence on the selective sensitivity of RUNX1mut blasts was also demonstrated in our previous study where a lymphoid-BP patient with a dominant RUNX1mut clone (VAF:48%) received a DSRT-based VEGFR inhibitor axitinib, which yielded clearance of the RUNX1mut clone at relapse [Development of new therapeutic options is essential for management of BP-CML . We iden CBF-AML , 53. Lik CBF-AML  and BCL2 CBF-AML  showed iactivity , which p1mut AML . Combina relapse .RUNX1mut BP-CML patients with CD19 expression. RUNX1 mutations are associated with upregulation of several molecular targets for immunotherapy, including CD19 [RUNX1mut BP-CML blasts in both myeloid-BP and lymphoid-BP patients. In combination with imatinib, CD19-CAR T cells showed enhanced killing of RUNX1mut BP-CML blasts. CD19-CAR T cells successfully targeted imatinib-resistant blasts, highlighting CD19-CAR T cells as a potentially effective strategy in BP-CML specially CD19-positive RUNX1mut BP-CML patients. The therapeutic approach combining TKIs and CD19-CAR T cells may also reduce the possibility of CD19-neg relapses, previously encountered both with CD19 targeting antibodies and CD19-CAR T cells in ALL [RUNX1mut leukemia.Our study also highlighted immunotherapy as an effective approach for BP-CML management, especially in ing CD19  and CD13ing CD19 . CD19-CAing CD19 . Combinaing CD19 , 60. We s in ALL , 62. Ints in ALL  suggestiRUNX1 mutations in CML progression by induced transcriptional reprogramming and aberrant mutagenic AID/RAG activity. Employing comprehensive phenotypic, genetic, transcriptional, and drug sensitivity profiling data highlighted multiple deregulated signaling pathways that represent novel options for targeted therapy, and together with CD19-CAR T-cell immunotherapeutic approach, may provide a means to improve management of poor prognosis BP-CML patients.In conclusion, this study provides insights into the role of Supplemental materialSupplemental dataset 1Supplemental dataset 2Supplemental dataset 3Supplemental dataset 4Supplemental dataset 5Supplemental dataset 6Supplemental dataset 7Supplemental dataset 8Supplemental dataset 9Supplemental dataset 10


Despite routine performance in patients who had received 223Ra, it is not clear whether disease sites refractory to alpha radiation can be effectively treated with 177Lu-PSMA-617. Secondly, it remains to be elucidated if therapy with 177Lu-PSMA-617 can be safely performed shortly after 223Ra, bearing in mind the myelotoxic potential of both treatments. The aim of our retrospective study was to evaluate safety and efficacy of 177Lu-PSMA-617 within less than 8 weeks after failing 223Ra. Radioligand therapy with 177Lu-PSMA-617 shortly after failing 223Ra is effective and can result in long-standing disease control even in patients with disseminated or diffuse bone involvement. Patients with oligo- and multifocal bone metastases show significantly longer overall survival with lower risk of significant hematotoxicity compared to patients with disseminated/diffuse involvement.The alpha emitter 223Radium-dichloride (223Ra) is an established treatment prolonging survival and reducing morbidity in selected patients with metastatic castration-resistant prostate cancer (mCRPC) with skeletal involvement. Radioligand therapy with 177Lutetium-PSMA-617 (177Lu-PSMA-617) has been increasingly implemented in patients with mCRPC failing conventional treatment options. In this study, the safety and efficacy of 177Lu-PSMA-617 in patients with progressive bone involvement under treatment with 223Ra was assessed. Twenty-eight men  with progressive mCRPC, who started 177Lu-PSMA-617 within 8 weeks after the last 223Ra administration, received a median of 4 (IQR 3–6) and a total of 120 cycles of 223Ra and a median of 4 (IQR 2–7) cycles 177Lu-PSMA-617 with a mean treatment activity of 6.5 ± 1.2 GBq per cycle, reaching a mean cumulative activity of 30.7 ± 23.4 GBq. A PSA response (≥50% PSA decline 12 weeks after the first 177Lu-PSMA-617 cycle) was observed in 18/28 (64.3%) patients and imaging-based partial remission (PR) was observed in 11/28 (39.3%) patients. Median imaging-based progression-free survival (PFS) was 10  months and median overall survival (OS) was 18  months. Patients with low bone tumor burden (2–20 lesions) had a significantly longer OS  compared to patients with a high tumor burden (>20 lesions). Grade ≥ 3 hematological toxicity was observed in six patients after their last treatment cycle with anemia, leukopenia and thrombocytopenia in 5/28 (17.9%), 4/28 (14.3%) and 6/28 (21.4%) patients, respectively. In progressive bone-metastatic mCRPC patients, prompt initiation of 177Lu-PSMA-617 after failing 223Ra is effective with an acceptable toxicity profile.Bone-seeking  Bone  (223Ra) ,5,6,7.223Ra is an established treatment prolonging survival and reducing morbidity in patients with osteoblastic bone dominant disease and no visceral metastases [177Lutetium (177Lu) allows selective radiation of PSMA-expressing cells, whilst sparing surrounding normal tissue, regardless of metastatic site.Radionuclide therapy with the bone-seeking alpha emitter tastases . Radiolitastases ,10. PSMA177Lutetium-PSMA-617 (177Lu-PSMA-617) included patients previously treated with 223Ra and few studies reported the safety of 177Lu-PSMA-617 in such patients [177Lu-PSMA-617 directly after failure of 223Ra. It is not yet clear if bone metastases refractory to 223Ra can be effectively treated with 177Lu-PSMA-617. Furthermore, the safety of 177Lu-PSMA-617 immediately after 223Ra needs to be investigated, considering the myelotoxic potential of both treatments. This study aims to assess the safety and efficacy of 177Lu-PSMA-617 of patients with progressive bone involvement under treatment with 223Ra starting within 8 weeks after the last 223Ra application.Several published studies on RLT with patients ,12,13. H177Lu-PSMA-617 within 8 weeks after the last 223Ra administration, were included in this retrospective study. Progression was assessed by bone scan routinely performed after the 3rd and the 6th cycle or in case of biochemical progression. A median of 4 (IQR 3–6) and a total of 120 cycles of 223Ra were administered with a mean activity of 4.197 ± 0.590 MBq, and no acute renal or bone marrow toxicities were observed. Other previous treatments comprised androgen receptor signaling inhibitors  and taxane-based chemotherapy  with progressive mCRPC, who started otherapy .177Lu-PSMA-617 included an estimated glomerular filtration rate (eGRF) of ≥30 mL/min/1.73 m2, hemoglobin (Hb) ≥ 8 g/dL, a white blood cell count (WBC) ≥ 2000/µL, platelets (Plt) ≥ 75,000/µL and sufficient PSMA-expression on 68Ga-PSMA-11 PET/CT imaging. The extent was categorized into (1) low  and (2) high . Written informed consent was given prior to each treatment cycle. Patient characteristics are depicted in Treatment initiation was approved by an interdisciplinary tumor board. General prerequisites for 177Lu-PSMA-617. PSMA-617 ligand was supplied by ABX . Labeling was performed in-house with 177LuCl3  [Inpatient treatments were carried out every 6–8 weeks using standard activities of 6–7.4 GBq Germany) .PSMA-based imaging was performed every 2–3 cycles for imaging based response assessment according to a recent consensus , as follBlood values , PSA and renal function based on eGFR were tested prior to each cycle, every 4 weeks throughout the treatment and every 6–12 weeks after the last cycle. Common Terminology Criteria for Adverse Events (CTCAE), version 5.0 was used to grade hematologic adverse events. Grade ≥ 3 toxicities were denominated significant.p < 0.05. Continuous variables are presented as median with interquartile range (IQR) or mean ± standard deviation. Categorical variables are shown as frequencies and percentages. Progression-free survival (PFS) is defined as the time interval from 177Lu-PSMA-617 initiation until progression on molecular, PSMA-based imaging, or the onset of other systemic treatments. Overall survival (OS) is defined as the time between the initiation of radioligand therapy and death from any cause; censoring was performed if the patient was alive at the time of analysis. The Kaplan–Meier method was used to calculate PFS and OS. Cox regression analysis was used to test the association of treatment response and survival.Retrospective data analysis was approved by the local ethics committee and was carried out using SPSS . The significance level of all tests was set at 223Ra, radioligand therapy (RLT) with 177Lu-PSMA-617 was initiated within 5 ± 3 weeks after the last 223Ra administration. Three patients developed visceral metastases under 223Ra . Upon initiation of 177Lu-PSMA-617, 16 patients had low  and 12 patients had high  bone tumor burden (based on PROMISE miTNM and previous studies) [For 28 patients showing progressive bone-metastatic disease under studies) ,19.177Lu-PSMA-617 with a mean activity of 6.5 ± 1.2 GBq per cycle, reaching a mean cumulative activity of 30.7 ± 23.4 GBq. A PSA decline of ≥50% 12 weeks after the first treatment cycle was seen in 18/28 (64.3%) patients, 7/28 (25.0%) patients showed a >25% PSA increase.A total of 131 treatment cycles were performed at intervals of 6 to 8 weeks. Patients received a median of four cycles (IQR 2–5) 177Lu-PSMA-617 consisted of PR in 11/28 (39.3%) patients, SD in 8/28 (28.6%) patients, and PD in 9/28 (32.1%) patients. An example of the disease course on 68Ga-PSMA-11 PET/CT imaging is provided in Imaging-based response to p = 0.001) as well as longer OS  (p = 0.589) and OS  (The median PFS for the whole study cohort was 10 (95% CI 6–14) months and the OS was 18 (95% CI 14–22) months a. PSA-re= 0.047) c. In con= 0.894) b.p < 0.001) months (p < 0.001) and PFS . Patients with disseminated/diffuse bone involvement at baseline (n = 12) showed a significantly shorter OS , despite similar PFS  compared to patients with oligo-/multifocal bone metastases (n = 16)  months than patients with SD (95% CI 8–28) 18 months and PD 11  months c. Diseas(n = 16) d.p = 0.026).In total, 17 out of 28 (60.7%) patients received subsequent therapies upon progression , 13 of wAt baseline, 24/28 (85.6%) patients had low grade anemia, 7/28 (25.0%) leukopenia and 4/28 (14.3%) thrombocytopenia .177Lu-PSMA-617. Mean Hb changed from 12.1 ± 1.4 to 10.1 ± 2.1 g/dL (p = 0.001), WBC from 5.2 ± 1.9 to 4.0 ± 1.6 × 109/L (p < 0.001), and Plt from 189 ± 51 to 125 ± 74 × 109/L (p = 0.011)  .177Lu-PSMA-617 cycle with anemia in 5 (17.9%), leukopenia in 4 (14.3%) and thrombocytopenia in 6 (21.4%) necessitating transfusion therapies. Overall, 5 out of 6 had high bone tumor burden at baseline.Overall, 6 out of 28 (21.4%) patients developed significant hematotoxicity (grade ≥ 3) after their last All six patients with significant hematotoxicity also showed disease progression which resulted in treatment discontinuation. One out of six patients received abiraterone and re-treatment with enzalutamide and lived 11 months after the last cycle. Another patient received docetaxel and repeated transfusion therapy and lived 13 months. All other patients died within 4 months after the last treatment cycle. Characteristics of these patients are detailed in At baseline, 15/28 (53.6%) patients showed low grade renal function impairment (grade 1/2). Moderate function decline to grade 2 was seen in 7/28 (25.0%) patients but no significant nephrotoxicity occurred.223Radium-dichloride and PSMA-targeting radioligand 177Lutetium-PSMA-617 is the selective internal radiation of prostate cancer cells. Despite being a common scenario in clinical practice, only few studies focused on the outcome of 177Lu-PSMA-617 in patients previously treated with 223Ra and no report exists on the efficacy of RLT with 177Lu-PSMA-617 in patients refractory to 223Ra. The observed PFS of 10 months and OS of 18 months in this retrospective study demonstrate efficacy of RLT with 177Lu-PSMA-617 in mCRPC patients failing 223Ra with progressive bone metastases.The rationale of systemic treatments with the bone-seeking calcium mimetic 177Lu-PSMA-617 in patients with mCRPC [177Lu-PSMA-617 when added to standard care in patients with progressive mCRPC after exhausting at least one taxane-based chemotherapy regimen and one androgen-receptor-pathway inhibitor [177Lu-PSMA-617 and standard care had a longer PFS  and OS  as compared to standard care alone. The influence of resistance to 223Ra was not evaluated as patients receiving 223Ra within 6 months of randomization were excluded.Following several retrospective studies on the value of th mCRPC ,20, the nhibitor . Patient177Lu-PSMA-617 may not be affected by previous exposure to 223Ra but it is unclear if 177Lu-PSMA-617 could produce any objective response in patients failing 223Ra. In a recent study by Sartor et al., 26 patients receiving 1 to 6 (median 6) 223Ra administrations 1 to 31 months (median 8 months) before the start of 177Lu-PSMA-617 were analyzed [223Ra and 177Lu-PSMA-617. An OS of 13.2 (95% CI 8.4–16.2) months was reported but no information on response to 177Lu-PSMA-617 and PFS was given. In another multicenter study (WARMTH), 223Ra 1–36.4 months before the first cycle of 177Lu-PSMA-617 had no impact on OS  [223Ra in some patients, but no information was given regarding the outcome of 177Lu-PSMA-617 in patients failing 223Ra. No further information was provided regarding the outcome of 177Lu-PSMA in this subgroup. In our study, RLT with 177Lu-PSMA-617 starting within 8 weeks from 223Ra could yield long-lasting disease control (PR + SD) in 19/28 (67.9%) patients with a PFS of 12 (95% CI 11–13) months. This finding indicates the efficacy of 177Lu-PSMA-617 in patients with progressive bone-metastatic mCRPC irrespective of recent resistance to 223Ra. Disease control was also associated with a significantly longer OS . The relatively long OS even in non-responders may be due to subsequent treatments after 177Lu-PSMA-617 in most patients (n = 5/9) with a significant life-prolonging effect.Few reports suggested that survival outcome of patients undergoing analyzed . Thirtee = 0.34) . The med177Lu-PSMA-617 [p = 0.001; OS22 vs. 15 months, p = 0.047). The PSA response rate of 64.3% in our cohort is in line with results from the phase II TheraP trial showing ≥50% PSA decline after 177Lu-PSMA-617 in 66% (65/99) of mCRPC patients previously treated with docetaxel; no information regarding previous treatment with 223Ra and PFS in PSA responders was reported [223Ra  compared to patients treated with 1 to 6 cycles 223Ra within up to one year (median: 11 weeks) before the start of 177Lu-PSMA-617 . The significance of initial PSA response (≥50% after 4 weeks) to 177Lu-PSMA-617 as a positive predictor of outcome remains controversial. A few studies in heterogeneous patient cohorts showed a longer OS in patients with early PSA response [223Ra, a PSA decline of ≥ 50% was associated with a significantly longer OS  [n = 14/28, 50.0%) with PSA response after the first cycle showed only a trend towards longer imaging-based PFS .PSA response, defined as ≥50% PSA reduction at 12 weeks, has also been used in other studies for treatment response assessment in patients receiving PSMA-617 ,23. In oreported . In a stresponse ,21,25, wa n = 17/; 58.6% c 0.0006) . In our 223Ra has a relatively low myelosuppressive potential, possibly owing to the short range (<100 μm) of high-energy alpha particles and its quick blood extraction [177Lu-PSMA-617 has a comparably low toxicity profile due to direct binding to the cell membrane and internalization, sparing the surrounding tissue [223Ra to the kidneys is extremely low, as the major route of elimination is the bowel. 177Lu-PSMA-617 has demonstrated low nephrotoxicity due to its rapid renal excretion. In our patients, no grade ≥ 3 nephrotoxicity occurred.traction . In the g tissue . Radiati177Lu-PSMA-617 [223Ra was not significantly associated with occurrence of hematotoxicity . In our collective of patients receiving 177Lu-PSMA-617 immediately after 223Ra, significant hematotoxicity occurred more frequently (21.4%). However, this may be only partially explained by the short time interval between the two treatments. In a preliminary study, Sartor et al. observed similarly high rate of hematotoxicity under 177Lu-PSMA-617 in patients treated with 223Ra a median of 8 months before starting 177Lu-PSMA-617 [177Lu-PSMA-617, especially grade 3 anemia. The authors suggested increasing tumor load as an underlying factor.Previous studies reported grade ≥ 3 hematologic adverse events in the range of 8–24% for anemia, 0–8% for leukopenia and 1–11% for thrombocytopenia ,24,28,29PSMA-617 . Here, p177Lu-PSMA-617. Groener et al. showed, that patients with disseminated/diffuse osseous involvement at baseline (n = 77/140) had a significantly higher incidence of hematotoxicity  [n = 18), no significant leukopenia was observed [n = 12/28) also showed a significantly shorter OS compared to patients with low bone tumor load (p = 0.045) despite similar PFS (p = 0.701).High extent of metastatic bone involvement may increase the risk of myelotoxicity and is suggested as a negative prognostic factor for survival after RLT with  = 0.04) . Similarobserved . In our . Firstly, similar to previous studies, using standard activities for all patients may be considered as a confounding factor in the evaluation of response to treatment and survival outcome. The retrospective nature of our study and the small cohort inevitably limit the strength of the results and impede extensive subgroup analysis. Moreover, patients with temporary myelotoxicity of grade ≥ 3 who recovered later than 8 weeks after the last 223Ra treatment were not included in this analysis. Subsequent therapies contributed to longer OS; therefore, PFS may be a more appropriate surrogate for evaluation of treatment efficacy in our study.Our study has clear limitations177Lu-PSMA-617 can be effectively and safely initiated as early as 8 weeks after failure of 223Ra in patients with progressive bone-metastatic mCRPC refractory to 223Ra. Objective response can be achieved even in patients with more advanced disease and disseminated/diffuse bone metastases, albeit with increasing incidence of significant hematotoxicity.RLT with


Scientific Reports 10.1038/s41598-020-64061-w, published online 29 April 2020Correction to: The Supplementary Tables 3S and 4S published with this Article contain errors, where data for the SNP profiles was coded incorrectly.The correct Supplementary Information tables are linked to this correction notice.Supplementary Table 3S.Supplementary Table 4S.


Trp metabolite L-Kyn promoted Breg differentiation in-vitro in an aryl hydrocarbon receptor (AhR), toll-like receptor-4-myeloid differentiation primary response 88, (TLR4-MyD88) dependent manner. Importantly, using mouse models with conditional deletion of IDO in myeloid-lineage cells, we identified a significant role for immunosuppressive myeloid-derived suppressor cell (MDSC)-associated IDO in modulating in-vivo and ex-vivo differentiation of Breg. Our studies thus identify Trp metabolism as a therapeutic target to modulate regulatory B cell function during lung cancer progression.Regulatory B cells (Breg) are IL-10 producing subsets of B cells that contribute to immunosuppression in the tumor microenvironment (TME). Breg are elevated in patients with lung cancer; however, the mechanisms underlying Breg development and their function in lung cancer have not been adequately elucidated. Herein, we report a novel role for Indoleamine 2, 3- dioxygenase (IDO), a metabolic enzyme that degrades tryptophan (Trp) and the Trp metabolite L-kynurenine (L-Kyn) in the regulation of Breg differentiation in the lung TME. Using a syngeneic mouse model of lung cancer, we report that Breg frequencies significantly increased during tumor progression in the lung TME and secondary lymphoid organs, while Breg were reduced in tumor-bearing IDO deficient mice (IDO Lung Cancer is the leading cause of cancer-associated morbidity and mortality among men and women, claiming ~1.3 million deaths annually worldwide (The tumor microenvironment (TME) plays a dynamic role in regulating immune responses during the progression of lung cancer , 5. TumoThe significance of Bregs in anti-tumor immunity became clear when improved therapeutic efficacy was observed in lung tumor-bearing B cell-deficient mice following treatment with a combination of chemotherapy and IL-15 . A signivia a specific lineage factor. Thus, investigating novel mechanisms of regulation of Breg will lead to the identification of promising targets for cancer therapeutics.Lineage-specific transcription factors have so far not been identified for Breg, suggesting that Breg are reactive rather than lineage specific. It is well known that immature B cells, mature B cells and plasmablasts may differentiate into IL-10 producing Breg through activation of Toll-like receptor (TLR) and CD40 , 31. Imm+ T cells and the expansion of Treg  mice suggesting an important role for IDO pathway in Breg infiltration and differentiation. Further, using mice with conditional deletion of IDO in myeloid lineage cells, we provide evidence that MDSC-associated IDO contribute significantly to Breg infiltration during lung cancer progression. Additionally, in-vivo and ex-vivo studies with AhR deficient mice (AhR-/-) indicate a potential role for AhR in Breg differentiation and corroborate our hypothesis that L-Kyn induced differentiation of Breg precursors is AhR independent whereas IL-10 producing Breg require AhR signaling. In addition, our ex-vivo data generated from TLR-4, MyD88, TLR-2, and TLR-2, 4 deficient B cells suggest a crucial role of TLR-4-MyD88 signaling in L-Kyn induced Breg differentiation. Collectively, our present study reveals a novel role of IDO and the Trp metabolite L-Kyn in Breg differentiation during the progression of lung cancer.In this study, we provide direct evidence for Trp-metabolite L-Kyn in modulating differentiation of Breg 6 LLC cells per mouse in 100 μl PBS) at 70-80% confluency were injected via intracardiac (i.c.) or tail vein (i.v.) route into. C57BL/6 and B6129-Ido1/J (IDO-/-) mice . Tumor weights at early time points in IDO deficient mice are not accurately measurable and hence these measurements were made 9 days post tumor implantation as we have described before  (American Type Culture Collection (ATCC) ) were propagated in Dulbecco’s Modified Eagle Medium (DMEM) with supplementation of 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 μg/ml penicillin-streptomycin and 0.1 mM non-essential amino acids . LLCs  were generated by inserting a 75bp loxP cassette 5’ of the second exon of the reference gene, NM_008324.2, by Ingenous targeting laboratory . A second loxP site was inserted 3’ of the fourth exon. Targeting was performed with C57BL/6 embryonic stem cells and microinjected into Balb/c blastocysts. Chimeras with a high percentage black coat color were mated to C57BL/6 FLP mice to remove the Neo cassette. Cre recombinase excises a 2.37 kb region of the Ido1 gene. Ido1M-Cre mice were generated by crossbreeding Ido1fl mice to M-Cre mice (B6.129P2-Lyz2tm1(cre)Ifo/J; JAX 004781) to target Ido1 inactivation in myeloid-derived cells. Mice were bred to homozygosity for ease of colony propagation. Both male and female mice of 6-8 weeks old were used for st strand cDNA synthesis kit (cat# 6110A) from TaKaRa Biotechnology, USA, VeriQuest SYBR Green qPCR master mix from Affymetrix, USA and Mouse IL-10 ELISA kit (cat# 88-7105-22) from ebioscience, USA.We used the following antibodies and reagents: anti-CD19 (clone: eBio1D3)-PE-Cy7, anti-CD5 (clone: 53-7.3)-APC, anti-CD1d (clone: 1B1)-eFluor 450, anti-IL-10 (clone: JES5-16E3)-PE, anti-Gr-1 (clone: RB6-8C5)-PE-Cy5, anti-CD11b (clone: M1/70)-APC-Cy7)-PE, anti-TCRαβ (clone: 145-2C11)-PE-Cy5,anti-IgM (clone: II/41)-FITC, anti-IgD (clone: 11-26C)-PerCPeFluor710, anti-B220 (clone: RA3-6B2)-APC-eFluor780, anti-CD21 (clone: eBio4E3)-FITC, anti-CD43 (clone: eBioR2/60)-PE, anti-CD23 (clone: B3B4)-PE, anti-CD93 (clone: AA4.1)-APC, anti-CD24 (clone: M1/69)-APC, and anti-IgM (clone II/41)-eFluor 450. All the antibodies were purchased from ebiosciences, USA. Anti-CD3 (clone: 145-2C11)-APC-Cy7 and the purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block™) (clone#2.4G2) was from BD Pharmingen, USA. Polyclonal AhR antibody (cat# BML-SA210-0025) was obtained from Enzo Life Sciences, USA. Histone H4 Antibody (cat# SC25260) and GAPDH Antibody (cat # SC365062) from Santa Cruz Biotechnology, USA. Anti-Rabbit IgG HRP conjugate (cat # W401B) and Anti-mouse IgG HRP conjugate (cat # W402B) from Promega, USA. L-Kyn (cat# K8625), Lipopolysaccharide (LPS) (cat# L4391), AhR antagonist CH-223191 (cat# C8124) and 1-Methyl-D-tryptophan (D-1MT) (cat# 452483) from Sigma-Aldrich. Recombinant murine- rIFN-ϒ (cat# 315-05) from PeproTech and CellTrace CFSE (cat# C34554) from invitrogen USA. Easy-Sep™ Mouse B Cell Isolation Kit (cat# 19854) from Stem Cell Technology, USA, Cell fractionation kit-Standard (cat# ab109719) from abcam, USA, Pierce BCA Protein Assay Kit (cat# 23225) from Thermo Scientific, USA, Pure-Link™ RNA Mini Kit (cat# 12183018A) from Invitrogen, USA, Prime script 16 cells were used for surface staining post Fc blocking (1μg/ml) in 3% BSA or FBS for 30 minutes over ice. For intracellular IL-10 staining 0.5-1×106 cells were cultured per well in 96-well plates  then incubated for 5hrs in stimulating RPMI media containing Phorbol 12-myristate 13-acetate, or PMA, , ionomycin , LPS  and a 1:1000 dilution of Golgistop/Golgiplug (BD Biosciences). Following stimulation, cells were first surface stained for 30 minutes at ice. After washing with PBS cells were fixed and permeabilized using the BD cytofix/cytoperm kit (BD Biosciences) for 30 minutes at 4°C then washed again with BD perm wash kit (BD Biosciences) and stained with intracellular fluorescently labeled anti-IL-10 antibodies. Fluorescence intensity of fluorochrome-labeled cells was measured by flow cytometry (BD LSR-II) while required sorting was done by BD FACS Aria III cytometer (UAB Flow Cytometry Core Facility). FACS Diva was used for acquiring the cells, and final data analysis was performed by Flow Jo . We included the whole nuclear cell population, including the tumor cells (CD45neg) for the analyses of % total Breg.Spleens were isolated from mice and macerated in RPMI 1640  supplemented with 10% FBS to prepare a single-cell suspension. Isolated tumor tissues were first macerated in serum-free Iscove’s Dulbecco’s Modified Eagle Medium (IDMEM) (Corning) and then digested in serum-free IDMEM contained with 2 mg/ml of collagenase B (Roche) and 0.02 mg/ml of deoxyribonuclease from bovine pancreas (Sigma) at 37°C for 30 min. After digestion, suspensions were neutralized with sterile IDMEM supplemented with 10% FBS filtered through 0.45 mm strainers. Red blood cells (RBCs) were lysed with ACK lysis buffer  incubated at room temperature for 1 minute and washed with 10% RPMI 1640. The cells were counted, and 0.5-1×108/ml splenocytes were resuspended in RoboSep buffer  followed by the addition of rat serum (50µl/ml)  to the sample. Samples were transferred then to 5ml polystyrene round-bottom tube and B cell isolation Antibodies cocktail (50µl/ml) was added and mixed by pipetting, then incubated for 10 minutes at room temperature (RT). Rapid sphere (75µl/ml)  was then added to the sample after vortexing it for 30 sec then mixed and incubated for 2.5 minutes at RT. RoboSep buffer was topped up to sample to make total volume 2.5ml and pipetted gently. Sample tubes were then placed in magnet  and incubated for 2.5 minutes at RT. Magnet was picked up and inverted the magnet tube pouring off the enriched cell suspension into a new tube. Purified B cells were washed twice with 10% RPMI 1640 and counted. B cells were disaggregated and stained with CFSE if needed and then 5 x105 cells were seeded in 200 µL per well in a 96 round bottom well plate with LPS (10μg/ml), increasing concentrations of L-Kyn (50µM and 100μM) and LPS + L-Kyn + CH 223191(10μM) an aryl hydrocarbon receptor antagonist (AhRA) for 72 hrs. Bregs were identified as CD19+CD5+CD1dhiIL-10+ by flow cytometry.Spleens or Bone marrow (BM) were isolated from mice and macerated in RPMI 1640  supplemented with 10% FBS to prepare a single-cell suspension. Red blood cells (RBCs) were lysed with ACK lysis buffer , then washed with 10% RPMI 1640. Cells were rewashed again in 1x PBS, and B cells were purified following manufacturer’s protocol. Briefly, 1x10in-vitro co-culture of B cells and MDSCs, CD19+ B cells were purified from splenocytes of naïve mice and co-cultured in a 5:1 ratio with MDSCs (CD11b+Gr-1+) sorted from the spleens and tumors of tumor-bearing mice and incubated for 72 hrs. For the in-vitro co-culture of B and T cells, T cells (CD3+TCRαβ+) were sorted from splenocytes of naïve mice and co-cultured in 1:1 ratio with purified B cells and incubated for 72 hrs. Bregs were identified in the co-culture as CD19+CD5+CD1dhiIL-10+ by flow cytometry.For the ex-vivo experiments following stimulation with LPS and LPS+L-Kyn using protocols provided with the Cell fractionation kit-Standard . Protein concentrations were measured using Pierce BCA Protein Assay Kit . 20-40µg of the cytoplasmic and nucleoplasmic fractions were used for western blot analysis to detect AhR expression on 10% SDS-PAGE gels. Gels were transferred overnight on to 0.45 μm Immobilon-P PVDF membranes (EMD Millipore). Blots were blocked in 5% non-fat dry milk (Lab Scientific) for 1 hour and incubated with primary antibody (manufacturer’s recommended concentrations) overnight at 4°C and then incubated in secondary anti-rabbit or anti-mouse HRP-conjugated antibodies , and protein bands detected using chemiluminescence .Cytoplasmic and nucleoplasmic fractions were extracted from cell pellets collected from ex-vivo experiments following stimulation with LPS, LPS+L-Kyn, LPS+L-Kyn+AhRA, and LPS+AhRA using Pure-Link™ RNA Mini Kit  and by following manufacturer’s recommendations. cDNA was made using Prime script 1st strand cDNA synthesis kit . Reverse transcription was conducted with Veri Quest SYBR Green qPCR master mix . The primer sequences used were as follows: AhR , Cyp1A1  and RPS-18 was selected as reference or housekeeping genes .Total RNA was isolated from cell pellets collected from +B220+ B cells from tumor-bearing WT or IDO deficient mice were labeled with CFSE  and culture for 4 days with 20 ug/ml LPS  and 10 ng/ml IL-4 . The percentages of CD19+CFSElow cells (proliferating cells) were determined by FACS analysis.Splenocytes or sorted CD19The mouse sera were collected from naive and tumor-bearing mice. The levels of IL-10 were determined by ELISA kits from ebioecience in sera following the manufacturer protocol. The supernatants from splenocytes or purified B cells from tumor-bearing WT or IDO deficient mice were collected on day 4 of culture. The levels of IgG were determined by ELISA kit from Life Technologies  in the supernatants following the instruction of the manufacturer.Statistical analyses were performed using Graph pad prism 5 software  and values were presented as mean ± SD unless indicated otherwise in the figure legend. Significant differences between the group means were determined by One-way ANOVA with Tukey’s multiple comparisons test for multiple groups. Comparisons between two groups were evaluated by an unpaired parametric student’s t-test. A value of p<0.05 was considered statistically significant.+CD1dhiCD5+IL-10+ Bregs which significantly increased in a time-dependent manner . We and others have previously reported that immunosuppressive MDSCs are important drivers of tumor growth in this tumor model (-/-) and wild type C57BL/6 (WT) control mice. Significantly reduced Breg frequency was observed in spleens and tumors of IDO-/- compared to WT control mice at 9 days following in-vivo establishment of lung tumors .We first investigated if Breg play an important role in the progression of lung cancer. Using a syngeneic murine model of lung cancer with intravenous implant LLCs, we first studied the kinetics of infiltration of CD19or model , 45. Addor model . Recent or model , 49. Thet manner . We and t manner . We and . We then examined a potential direct role for MDSC-associated IDO as a driver of Breg differentiation in co-cultures of immune sorted MDSCs (CD11b+Gr-1+) from splenocytes and tumors of tumor implanted WT and IDO-/- mice with CD19+ purified B cells in 5:1 ratio (5 B cells: MDSC) . Impairment of Breg differentiation was observed in B cells co-cultured with MDSCs from IDO-/- compared to WT mice . Breg differentiation was significantly reduced when MDSCs (CD11b+Gr-1+) sorted from splenocytes of tumor-bearing IDO-/- mice were co-cultured with total splenocytes (5:1 ratio) compared to WT . Further, we validated the contribution of MDSC-associated IDO in Breg differentiation using IDO f MCre mice with conditional deletion of IDO in myeloid-lineage cells  and their MCre control mice. At day-9 post tumor implant, IDO f MCre mice exhibited significantly lowered tumor burden compared to controls . Importantly, we observed a significant reduction in Breg in tumor-bearing IDO f MCre compared to their MCre control . Collectively, these data suggest that Trp catabolizing enzyme IDO plays a major role in Breg differentiation in the lung TME with a significant contribution from MDSC-associated IDO.The immunosuppressive properties of Bregs have been attributed to IL-10 , 50, 51.. We then. We then. We then. We then. We then. We then. We thenex-vivo cultures of CD19+ B cells purified from splenocytes of naïve WT mice stimulated with no LPS controls and LPS+Kyn and LPS+Kyn+AhRA  treatments, we observed induction of CD19+CD5+CD1dhiIL-10+ Bregs frequency in response to stimulation with LPS+L-Kyn while Breg differentiation was inhibited with the addition of AhRA . In addition, we detected elevated levels of IL-10 in the culture supernatant from LPS+Kyn treated samples . These data suggest that L-Kyn promotes Breg through AhR pathway. Furthermore, CD19+CD5+CD1dhi Breg precursors were elevated following treatment with LPS+L-Kyn in a dose-dependent manner. But the expansion of these Breg precursors was present even in the presence of AhRA compared to LPS only stimulation ex-vivo, demonstrating that L-Kyn induced Breg precursor differentiation was AhR independent. We then evaluated the role of L-Kyn alone without LPS, and found that Breg precursors (CD19+CD5+CD1dhi) were enhanced, but L-Kyn alone was unable to transform them into IL-10 producing Breg, suggesting that LPS stimulation was essential to induce Breg precursors to become IL-10 producing Breg ex-vivo. Our qRT-PCR data validated this finding . TheAhR-Arnt heterodimers then finally bind with dioxin-responsive elements (DREs) to regulate gene expression in a variety of cells  in AhR deficient CD19+ B cells upon stimulation with LPS+L-Kyn compared to LPS only stimulation , strongly supporting our hypothesis that L-Kyn induced Breg precursor differentiation is AhR independent.Interestingly LPS+ L-Kyn was unable to induce Breg differentiation in AhRrol mice . Furtherrol mice . Furtherrol mice . Further+IgM+IgD+) upon stimulation with LPS+L-Kyn induced frequency of CD19+CD5+CD1dhi Breg precursors . Collectively these findings suggest that L-Kyn, an IDO-derived Trp metabolite activates AhR and translocate AhR complex into the nucleus of B cells to drive their differentiation into IL-10, producing Breg. At the same time, L-Kyn alone enhances the Breg precursor pool ex-vivo, which upon stimulation with LPS transform them into IL10 producing Bregs.CD40,TLRs, or cytokine-mediated activation turns immature B cells directly into Breg or immature B cells may first differentiate into mature B cells and then into IL-10 producing Breg . Therefovia transcription factor NF- κB to regulate several genes associated with the expression of antigen-presenting molecules, cytokines, transcription factors and enzymes including IDO  mice (+CD5+CD1dhiIL-10+) was analyzed in ex-vivo experiments performed with negatively selected purified B cells from WT, MYD88-/- and TLR2-/- following stimulation with high concentration of L-Kyn (100μM) alone compared to LPS alone. No significant change in Breg was noted among WT, MYD88-/- and TLR2-/- in absence of LPS . While the Breg precursors at baseline were significantly higher in the TLR-2,4-/- deficient mice compared to the individual KO, this did not correlate with baseline B reg numbers .Previously, LPS induced TLR-2 activation and TLR-2 dependent Breg induction have been reported , 57. To mulation . These dmulation . These dmulation . These d+ cells, B220+IgD-IgM-CD24intCD43+ pro-B cells, B220+IgD-IgM- CD24hiCD43- pre-B and B220+IgD+IgM+ mature B cells were decreased in BM of tumor-bearing IDO deficient mice compared with naïve IDO deficient mice . Furthermore, both the percentages and numbers of B220+CD93+ immature B cells were increased, whereas B220+CD93-CD21intCD23+ follicular B cells were decreased in tumor-bearing IDO deficient mice compared with naïve IDO deficient mice . However, we observed no difference in total B cells, pro-B cells, pre-B cells, immature B cells and mature B cells in BM , as well as total B cells, immature B cells, marginal zone B cells and follicular B cells in spleens  between WT and IDO naïve mice or tumor-bearing mice. These results indicate that IDO deficiency does not affect B cells development in BM and spleen both in naïve tumor-free mice and tumor-bearing mice.We then investigated if the reduction in B reg differentiation is due to alteration of overall B cell differentiation resulting from IDO deficiency. As we had reported earlier that B cell differentiation is impaired during tumor growth in IDO sufficient mice , we evalent mice . Furtherent mice . Furtherent mice . Furtherin vitro, splenocytes or sorted CD19+B220+ B cells from WT or IDO-/- tumor-bearing mice were labelled with CFSE and cultured in the presence of LPS and IL-4. We observed that the percentages of CD19+CFSElow cells were increased in splenocytes  or purified B cells  from IDO mice compared with that from WT mice. IgG production in culture supernatants was elevated in activated splenocytes  and/or purified B cells  from IDO-/- mice compared with WT controls. These results suggest that IDO negatively regulates B cell proliferation and IgG production in tumor-bearing mice.It has been reported that B cell-intrinsic IDO regulates humoral immunity to T cell-independent antigens . To inveenocytes  or purifenocytes  or purifenocytes  or purifex-vivo. Further, we report an uncharacterized role for AhR in regulating L-Kyn induced Breg differentiation. Our ex-vivo data generated from TLR-2, TLR-4, TLR-2, 4, and MyD88 deficient B cells indicate that L-Kyn induces Breg differentiation through TLR-4-MyD88 pathway ex-vivo while L-Kyn promotes Breg precursors independent of TLR-2 and TLR-4 signaling. Furthermore, our ex-vivo B and T cells co-culture studies support our conclusion that L-Kyn induces Breg differentiation independent of T cells. Importantly, we provide evidence from ex-vivo experiments that tumor antigens modulated by IFN-γ induces IDO, which then promotes antigen-specific Breg differentiation through AhR pathway. Collectively, these data conclude that IDO, a Trp metabolizing enzyme, and L-Kyn, a major metabolite of Trp promote Breg differentiation through AhR and TLR-4-MyD88 pathways.In this study, we demonstrate that splenic Breg frequency increases with the progression of lung tumor growth in a syngeneic model of lung cancer. Additionally, we report for the first time that IDO associated with tumors and MDSCs promote Breg differentiation during lung cancer progression. Our investigations suggest that IDO-induced Trp metabolite L-Kyn mediates the development of Breg precursors that differentiate into IL-10 producing Breg upon stimulation with LPS regs, MDSCs, and pDCs and very recently reported also by macrophages (+ dendritic cells can impair antigen presentation from antigen-presenting cells (APCs) in the tumor-draining lymph nodes to recruited effector T cells. Furthermore, tumor cells that express IDO not only promote Treg differentiation but also disable effector T cell function  further support the positive regulation by MDSC- associated IDO in Breg differentiation in-vivo. Therefore our current studies and previous evidence are in resonance with our observations that IDO from MDSCs and tumor cells promote Breg during lung cancer progression.We and others have reported previously that in addition to tumor cells, immunosuppressive MDSCs are the major contributors of IDO, a rate-limiting enzyme of Trp catabolism in the TME , 72, 73. the TME . There i the TME –76. We r the TME . It is nimmunity . Interestes Breg . To the + T cells in TME  independent of TLR-2 and TLR-4 signaling but depends on TLR-4-MyD88 pathway to transform them into IL-10 producing Breg cells. Furthermore, our hypothesis is also strongly proven by another ex-vivo data showing LPS (TLR-4 ligand) is required to transform Breg precursors into Breg cells.Immune cell activation by LPS is mainly mediated by TLR-4 . TLR lig-/- mice, that we report here, is not due to overall reduction B cell development or differentiation. Although we and others  and by Trp metabolite L-Kyn of tumor , 90. Conof tumor . ConsistThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee the University of Alabama at Birmingham, AL, USA.ST, YW, JJ, KH, and JD designed the experiments. ST, YW, JJ, KH, and JS and performed the experiments. ST, YW, and JD prepared figures and supplementary material sections. ST, YW, and JD wrote the manuscript. ST, YW JJ, KH, JS, MA, VT, RM, SP, and JD reviewed and revised the manuscript. All the authors contributed to final data analysis, discussions, and manuscript preparation.This study was supported by institutional support through ACS– IRG-60-001-53-IRG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RNF213 encode dominant-negative alleles that globally impair ubiquitylation by RNF213-UBE2D2.MMD-associated SNPs of  RNF213, which encodes a 591-kD protein with AAA+ ATPase and RING E3 domains, are associated with a rare, autosomal dominant cerebrovascular disorder, moyamoya disease (MMD). MMD-associated SNPs primarily localize to the C-terminal region of RNF213, and some affect conserved residues in the RING domain. Although the autosomal dominant inheritance of MMD could most easily explained by RNF213 gain-of-function, the type of ubiquitylation catalyzed by RNF213 and the effects of MMD-associated SNPs on its E3 ligase activity have remained unclear. We found that RNF213 uses the E2-conjugating enzymes UBE2D2 and UBE2L3 to catalyze distinct ubiquitylation events. RNF213-UBED2 catalyzes K6 and, to a lesser extent, K48-dependent poly-ubiquitylation in vitro, whereas RNF213-UBE2L3 catalyzes K6-, K11-, and K48-dependent poly-ubiquitylation events. MMD-associated SNPs encode proteins with decreased E3 activity, and the most frequent MMD allele, RNF213R4810K, is a dominant-negative mutant that decreases ubiquitylation globally. By contrast, MMD-associated RNF213 SNPs do not affect ATPase activity. Our results suggest that decreased RNF213 E3 ligase activity is central to MMD pathogenesis.Single-nucleotide polymorphisms (SNPs) in  Moyamoya disease (MMD) is a rare, autosomal dominant disorder characterized by stenotic/occlusive lesions in the circle of Willis with surrounding abnormal blood vessels in the brain . These vRNF213 , indicating that additional genetic and/or environmental modifiers are required for pathogenesis (MMD risk is strongly associated with single nucleotide polymorphisms (SNPs) in RNF213 . On averK allele . Severalnicities . Althougogenesis .RNF213 encodes an ∼591-kD protein containing tandem AAA+ ATPase domains and a RING E3 domain  transfer from (an) E2 ubiquitin-conjugating enzyme(s) to lysine residues of E3-bound substrate(s), resulting in isopeptide bond formation . Mono-ubRNF213, which includes the RING and RZ domains   showed tRNF213 in HeLa Flp-In T-Rex cells, resulting in HeLa Flp-In T-Rex KO cells  3x Flag-tagged RNF213 upon doxycycline induction (WT cells were co-transfected with an expression construct for HA-UB (HA-tagged Ubiquitin) and siRNAs targeting UBE2D2 or UBE2L3  is a(the) physiological E2 for RNF213.We therefore evaluated the ability of these E2s to act in concert with RNF213 in a physiologic context. By using CRISPR/Cas9 technology, we generated a null (frameshift) mutant of KO cells . We thennduction . Finallyr UBE2L3 . Flag-RNr UBE2L3 , RNF213  qRT-PCR , reduced qRT-PCR . In addiOthers have reported that RNF213 ubiquitylation activity in vitro is mediated by UBE2N/V1  or UBE2LFinally, we assessed the type of ubiquitin linkage added by RNF213 with UBE2D2 or UBE2L3 using UB “add-back” mutants. A UB mutant lacking all lysine residues (K0) abrogated RNF213 auto-ubiquitylation with either E2 . Using UR4810K has impaired ATPase activity , 3x-Flag-RNF213I3999A (RING-impaired), and four major MMD SNPs: 3x-Flag-RNF213D4013N, 3x-Flag-RNF213D4014N, 3x-Flag-RNF213K4732T, and 3x-Flag-RNF213R4810K , a RING E3-defective mutant (I3999A), and the most prevalent MMD variant (R4810K), to interact with EGFP-tagged WT RNF213. The AAA+ ATPase mutant alters the glutamic acid residue in the first Walker B motif (E2488), preventing ATP hydrolysis  expression led to lower global ubiquitylation than the most prevalent, non-RING allele, RNF213R4810K. Therefore, MMD penetrance might reflect the extent to which RNF213 E3 ligase activity is attenuated by a given MMD SNP in cells.To explore further the role of RNF213 E3 ligase activity in MMD, we assessed the E3 ligase activity associated with SNPs of different penetrance in vivo . When reRNF213 or RNF213 mutants into (endogenous RNF213-replete) 293T and HeLa cells. A portion of each transfected cell population was treated with proteasomal (MG132) and lysosomal (chloroquine) inhibitors to block UB-mediated degradation. Consistent with our previous findings, over-expression of WT RNF213 resulted in an increased number of ubiquitylated proteins relative to control, vector-transfected cells . Similar to the dominant-negative effects of the RING-impaired and MMD-associated RNF213 mutants, RNF213-depleted 293T and HeLa cells had a global decrease in ubiquitylation , impair the E3 activity of other complex members.At first glance, the finding that RNF213gomerize , indicatgomerize . HoweverRnf213−/− or Rnf213R4828K/+ mice. Furthermore, ∼2% of the Japanese population have RNF213 SNPs, yet the incidence of MMD in Japan is as low as 0.53 per 100,000. Notably, incidence is higher in children and adolescents (5–14 yr old) and is ∼2-fold more common in females than in males. MMD also is associated with anemia, autoimmunity, infection/inflammation, and radiation. Together, these findings predict that genetic or environmental modifier(s) are required to trigger MMD in patients bearing RNF213 SNPs (and presumably in Rnf213−/− or Rnf213R4828K/+ mice). It will be important to identify such modifiers and determine how they affect RNF213 E3 activity or its substrates. A clue might come from considering biological differences between mice and fish, as Rnf213 morphants in zebrafish do show abnormal vascular growth , UBE2D2 (Cat. no. L-010383-00-0005), or UBE2L3 (Cat. no. L-010384-00-0005) (Dharmacon), were introduced into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific), as per the manufacturer’s instructions.HeLa Flp-In T-Rex, HeLa, and 293T cells were grown in DMEM with 10% FBS (Invitrogen), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C. HeLa Flp-In T-Rex were purchased from Thermo Fisher Scientific. HeLa and 293T cell lines were obtained from the ATCC, authenticated by STR testing, and assessed monthly for the absence of mycoplasma contamination . Transfections were carried out by using Fugene 6 (Promega), according to the manufacturer’s instructions. For transient transfection experiments, the amount of plasmid DNA was kept constant between conditions in every experiment by adding appropriate amounts of empty vector. Control siRNAs (Cat. no. D-001810-10-05) or siRNAs targeting RNF213-knockout HeLa Flp-In T-Rex cells were generated using CRISPR/Cas9 technology (RNF213 (ACAATGGCGTCGGCCTCGGA) was designed by using http://crispr.mit.edu and cloned into the BbsI site of pSpCas9 (BB)–2A–Puro . HeLa cells were transiently transfected with the PX459–sgRNF213-exon3 vector using FuGENE 6 Transfection Reagent (Promega). 48 h post-transfection, cells were diluted and plated in 96 well plate (1 cell/well). RNF213-knockout clones were identified by DNA sequencing and confirmed by immunoblotting. For generating isogenic HeLa Flp-In T-Rex knockout cells expressing WT RNF213 and RNF213 variants upon doxycycline induction, pcDNA5/FRT/TO-based constructs expressing 3x-Flag tagged RNF213 or RNF variants were co-transfected with pOG44, which directs constitutive expression of Flp recombinase. Cells with successful integration of 3x-Flag-RNF213 or its variants were selected by adding hygromycin B (200 μg/ml) at 48 h post-transfection. RNF213 expression was induced by adding 1 μg/ml doxycycline.chnology . BrieflyCells were lysed in lysis buffer . For whole cell lysate immunoblots, equal amounts of protein per sample were subjected to SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). For immunoprecipitations, lysates were incubated with appropriate antibody-agarose beads, as indicated, for 4 h on a rotator-mixer at 4°C. Beads were washed five times in lysis buffer and then incubated in sample buffer. Lysates and immunoprecipitates were resolved by modified SDS–PAGE on 3–8% Tris-acetate gels or 5% Tris-glycine gels. Gels were transferred in 1× transfer buffer, 2% methanol for 16 h at 25 V. The following antibodies were used: RNF213 , HA , GST , Flag , ERK2 , and GFP . Blots were quantified by acquisition software, Image Studio Lite, using an Odyssey Infrared imaging system (Li-Cor Biosciences). Effects on overall ubiquitylation in RNF213 or RNF213 variants. RNF213 was induced with doxycycline (1 μg/ml) and lysed in RIPA buffer. Lysates were subjected to immunoprecipitations with anti-FLAG M2 magnetic beads  for 3 h on a rotator-mixer at 4°C. Beads were collected at bottom of the tube using a magnetic stand, supernatants were discarded, and immunoprecipitants were washed five times in lysis buffer followed by elution with 50 μl of 100 ng/μl 3x-Flag peptide. Briefly, 0.1 μM of purified RNF213 was used for in vitro auto-ubiquitylation reaction in the presence of 50 μM HA-Ub (Boston Biochem), 100 nM E1 (Boston Biochem), 1 μM E2 (Boston Biochem), 10 mM Mg-ATP (Boston Biochem) for the times indicated. For Ubiquitin linkage analysis, a 2-h incubation and ∼0.25 μM of purified RNF213 was used in the presence of 100 μM Ub and its linkage-specific addback mutants (Boston Biochem), 100 nM E1 (Boston Biochem), 1 μM E2 (Boston Biochem), and 10 mM Mg-ATP (Boston Biochem). Total volume of the reaction was 25 μl. Reactions were terminated by adding SDS–PAGE sample buffer, and ubiquitin incorporation was analyzed by immunoblotting, as described above.Full-length RNF213 was purified from HeLa Flp-In T-Rex KO expressing 3x-Flag tagged wild-type RNF213 mutants were generated using by PCR or synthesized by Genewiz. Fragments containing the desired mutation were sequenced and cloned into wild-type RNF213 plasmids using Gibson assembly  was incubated in assay buffer containing ATPase substrate, developed at 25°C for 15–30 min, and A650 was measured using FlexStation 3 Multi-Mode Microplate Reader. For full-length RNF213 purification, HeLa Flp-In T-Rex KO expressing 3x-Flag tagged wild-type RNF213 or RNF213 variants were induced with doxycycline (1 μg/ml) and lysed in ice cold ATPase Assay Buffer provided with the ATPase assay kit. Lysates were subjected to immunoprecipitations with anti-FLAG M2 magnetic beads  for 3 h on a rotator-mixer at 4°C. Beads were collected at bottom of the tube using a magnetic stand, supernatants were discarded, and immunoprecipitates were washed five times in lysis buffer followed by elution with 50 μl of 100 ng/μl 3x-Flag peptide.ATPase assays using full-length RNF213 were carried out using the ATPase Activity Assay Kit  as per the manufacturer’s instructions. Briefly, purified RNF213  along with deubiquitylase inhibitor (+10 mM IAM and NEM), and lysates were analyzed by immunoblotting as described above.Where indicated, cells were transfected with empty pRK5 vector or pRK5 expressing HA-UB (HA-tagged ubiquitin) by using Fugene 6 (Promega), as per the manufacturer’s instructions. In some experiments, a portion of the transfected cells was treated with 10 μM MG132 (Tocris) and 50 μM chloroquine (Tocris). Cells were lysed in lysis buffer . cDNA synthesis was carried out using the RT2 first stand kit (QIAGEN) cDNA synthesis kit following the manufacturer’s protocol. qPCR then was carried out using Maxima SYBR Green Supermix (Life Technologies) in an Applied Biosystems StepOne Real-time PCR machine. 18S rRNA was used as the internal control for all samples. For human Sample sizes and statistical tests for each experiment are mentioned in the figure legends. All analyses and graphs were generated using GraphPad Prism 9.


KRAS) is the most common driver in NSCLC, and targeting oncogenic KRAS is a major challenge in the treatment of non-small-cell lung cancer (NSCLC). While several covalent KRAS G12C inhibitors have emerged as a novel anti-KRAS therapy, the development of combined therapies involving the targeting of oncogenic KRAS plus other targeted drugs is still required given the vast heterogeneity of KRAS-mutated tumors. In this review, we summarize the biological and immunological characteristics of oncogenic KRAS-driven NSCLC and the preclinical and clinical evidence for mutant KRAS-targeted therapies. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible therapeutic strategies to overcome this drug resistance.v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting KRAS is considerably important. The recent discovery of covalent KRAS G12C inhibitors offers hope for improving the prognosis of NSCLC patients, but the development of combination therapies corresponding to tumor characteristics is still required given the vast heterogeneity of KRAS-mutated NSCLC. In this review, we summarize the current understanding of KRAS mutations regarding the involvement of malignant transformation and describe the preclinical and clinical evidence for targeting KRAS-mutated NSCLC. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible combination treatment strategies to overcome this drug resistance.Recent advances in molecular biology and the resultant identification of driver oncogenes have achieved major progress in precision medicine for non-small-cell lung cancer (NSCLC). v-Ki-ras2 Kirsten rat sarcoma viral oncogene ( KRAS), which is one of the most common oncogenes in NSCLC, had long been an “undruggable target” despite extensive efforts. RAS proteins are small guanosine triphosphatase (GTP)-binding proteins. The RAS family consists of three members, HRAS, NRAS and KRAS. KRAS knockout in mice is lethal, but HRAS or NRAS knockout mice are viable and develop normally . Th. ThRAS gKRAS copy number alterations have been reported in NSCLC [KRAS frequently occurred, and MACI with KRAS copy number gain was related to increased RAS GTPase activity [KRAS MASI has been associated with an unfavorable prognosis in LUAD [KRAS copy number gain resulted in metabolic rewiring and increased malignancy [KRAS amplification was also shown to be a mechanism of resistance to EGFR-TKIs [KRAS copy number alterations enhance oncogenic KRAS activity, leading to treatment resistance and a worse prognosis in KRAS-mutated NSCLC.in NSCLC . A mutanactivity . KRAS MA in LUAD ,25,26. Ilignancy . KRAS amGFR-TKIs . These sKRAS mutations varies depending on the mutation subtype in NSCLC. For instance, G12C and G12D have higher affinities for binding to RALGDS and PI3K, respectively [KRAS G12C mutations exhibited a higher programmed death-ligand 1 (PD-L1) tumor proportion score and tumor mutational burden than those with KRAS non-G12C mutations [KRAS G12C or G12V mutations have a worse prognosis than those without these mutation subtypes [KRAS G12C mutations had significantly shorter survival times than those with KRAS non-G12C mutations [KRAS G12C seems to be an attractive therapeutic approach to improve the prognosis of NSCLC patients. In contrast, no significant difference in the overall survival of patients with metastatic NSCLC was found among different subtypes of KRAS mutations [KRAS-mutated tumors [KRAS mutation subtypes [The biological and clinical significance of ectively . A recenutations . From a subtypes . In anotutations . Therefoutations ,33. Thesd tumors ,35, in asubtypes ,36.KRAS-mutated NSCLCs into subgroups with distinct biological and immunological characteristics to predict therapeutic vulnerabilities. Skoulidis et al. conducted an integrative analysis of genomic, transcriptomic and proteomic data, and defined three subtypes of KRAS-mutated LUADs based on co-occurring genetic events: the KC subgroup was defined by CDKN2A/B inactivation and reduced TTF-1 expression, the KL subgroup was defined by LKB1 alterations, and the KP subgroup was defined by TP53 alterations [KEAP1 gene encoding Kelch-like ECH-associated protein 1, and expressed lower levels of immune markers, such as lower PD-L1 levels, whereas KP tumors showed higher levels of somatic mutations, inflammatory markers, and immune checkpoint effector molecules, including PD-L1 and programmed death 1 (PD-1), accompanied by increased T cell infiltration [KEAP1-inactivating mutation is one of the more frequently co-occurring genetic alterations in KRAS-mutant/LKB1-mutant LUADs [KEAP1 inactivation aberrantly activates the NRF2 pathway, conferring a more aggressive tumor phenotype [KEAP1 and LKB1, as well as the activation of the YAP1 and/or RSK-mTOR pathway, contribute to mutant KRAS independency in tumor cells, potentially causing therapeutic resistance to KRAS inhibitors [KRAS-mutated NSCLC.Omics data analyses have attempted to classify erations . KC tumoltration . As descnt LUADs ,38. KEAPnt LUADs , and KEAhenotype ,41,42. Thibitors . Thus, cKRAS-mutated NSCLC tumors have inflammatory characteristics; oncogenic KRAS induces several inflammatory cytokines and chemokines, including IL-6, IL-8, CXCL1 and CCL5, which influence the TME [KRAS mutations, and is involved in the development of lung cancer [KRAS-mutated NSCLC cells produce CCL5 [There is growing evidence that oncogenic KRAS is involved in tumor immune evasion by regulating the TME . KRAS-mu the TME . For ins the TME . Oncogen the TME . Elevate the TME . Anotherg cancer ,50,51. Muce CCL5 , which puce CCL5 .KRAS-mutated NSCLC tumors exhibit PD-L1 overexpression [KRAS mutations [NT5E, encoding CD73, as a gene upregulated by KRAS mutations in NSCLC cells [EGFR-mutated NSCLC tumors [EGFR-mutated NSCLC cells with CD73 knockdown, and that an anti-CD73 antibody suppressed tumor growth in immunocompetent mice [KRAS-mutated NSCLC tumors have the ability to evade immune responses by regulating inflammatory cytokines and chemokines, immune checkpoint molecules and Tregs. Thus, targeting mutant KRAS may be an optional approach to abolish tumor immune evasion, and combination treatments including KRAS G12C inhibitors and immune checkpoint inhibitors could be effective therapeutic strategies.Several studies have demonstrated that pression , which ipression ,56,57,58utations . In addiutations . Mutant utations . We prevLC cells . SimilarC tumors . They fuent mice . These oTP53, LKB1 or KEAP1 co-occurring with KRAS mutations affect the TME. In a preclinical study, the ectopic expression of LKB1 induced a strong increase in PD-L1 expression in human NSCLC cells harboring KRAS mutations, whereas LKB1 knockdown decreased PD-L1 in mouse lung fibroblasts transformed with active KRAS and HPV16 E6E7 [LKB1 mutations were negatively associated with PD-L1 expression in KRAS-mutated NSCLC tumors, whereas gene expression signatures associated with the infiltration of effector T cells and dendritic cells were reduced in LUAD tumors with LKB1 mutations and NRF2 activation [KRAS/TP53-comutated LUAD tumors manifested increased expressions of PD-1 and PD-L1, a high accumulation of CD8+ tumor-infiltrating lymphocytes and high mutational loads, and NSCLC patients with co-occurring KRAS/TP53 mutations showed a favorable clinical benefit with anti-PD-1 therapy [Genetic alterations of V16 E6E7 . LKB1 mutivation . Meanwhi therapy .+ T cell exhaustion in tumors, including PD-1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) [KRAS mutations [KRAS/LKB1/KEAP1 comutations. These observations suggest the therapeutic significance of anti-VEGF therapies in combination with immune checkpoint inhibitors in patients with NSCLC harboring KRAS mutations.Vascular endothelial growth factor (VEGF) is a key player in tumor-induced immunosuppression in the TME ,67,68. V(CTLA-4) . VEGF al(CTLA-4) ,71. The (CTLA-4) ,73 and t(CTLA-4) . Of noteutations . MoreoveKRAS G12C mutations demonstrated the encouraging safety and anticancer activity of sotorasib monotherapy [KRAS G12C-mutant NSCLC patients who had received standard therapies [KRAS G12C-mutated advanced or metastatic NSCLC patients who have received at least one prior systemic therapy. Considering that the standard first-line therapy for patients with advanced NSCLC is an anti-PD-1/L1 antibody in combination with platinum-based chemotherapy [KRAS G12C-mutated NSCLC patients generally receive sotorasib in the second-line setting after chemotherapy with immune checkpoint inhibitors. It is noteworthy that a subgroup of patients treated with prior anti-PD-1/L1 therapy but not platinum-based chemotherapy had a high objective response rate (69.2%) and a median overall survival time of 17.7 months [KRAS G12C-mutant NSCLC patients who were previously treated with chemotherapy and an anti-PD-1/L1 antibody, showed that 45% of the patients (23/51) achieved a partial response (PR) and 26 patients had stable disease (SD) [KRAS G12C and LKB1 coalterations. These results suggest that NSCLC patients are sensitized to immune checkpoint inhibitors by the KRAS G12C inhibitors through tumor immune microenvironment reconditioning, as shown in preclinical models [The recent discovery of covalent KRAS G12C inhibitors has changed “undruggable” oncogenic KRAS into a “druggable” target. In the KRAS G12C inactive (GDP-bound) form, the mutant cysteine residues adjacent to the switch II pocket are involved in the effector interaction . The binotherapy . Subsequherapies . Of the otherapy , it is p7 months . Similarase (SD) . In thisl models ,81.KRAS-mutated NSCLC, it was shown that co-occurring KRAS mutations, including the pairs G12D/G12V, G12D/G12C, G12V/G12C and G12D/G12S, can be found in the same NSCLC tumors [KRAS mutations uncovered that 53.5% of the patients had additional mutations, including MET amplifications (15.4%), ERBB2 amplifications (13.8%), PIK3CA mutations (3.2%), EGFR mutations (1.2%) and BRAF mutations (1.3%) [KRAS, EGFR, ALK, ROS1 and BRAF in 3774 samples from NSCLC patients, the frequencies of single KRAS mutations and concomitant KRAS mutations were 8.0% and 1.1%, respectively, indicating that approximately 12% of KRAS-mutated NSCLC tumors had other driver mutations [KRAS-mutated tumors.While targeted therapies induce a dramatic tumor response early in treatment, most tumors ultimately become resistant to targeted drugs, due to the intratumoral heterogeneity that results in increasing levels of preexisting resistant clones and tumor evolution favoring gain of resistance ,83. For C tumors . A next-s (1.3%) . In anotutations . Such geKRAS mutations and activated bypass pathways potentially cause resistance to covalent KRAS G12C inhibitors in NSCLC [KRAS mutations , high-level amplifications of the KRAS G12C allele and alterations in RAS-related pathways  were found in tissue biopsy samples and/or circulating tumor DNA obtained from KRAS G12C-mutant NSCLC patients who had acquired resistance to adagrasib [KRAS G12C mutants; G12C/R68S, G12C/H95D and G12C/Y96C double mutants mediated resistance to adagrasib, while only G12C/H95D retained sensitivity to sotorasib. Similarly, Tanaka et al. found 10 distinct mutations  that affect RAS-MAPK signaling in cell-free DNA obtained from a patient who had developed progressive disease after adagrasib monotherapy [KRAS G12C mutants revealed several secondary KRAS mutations causing resistance to sotorasib and adagrasib [KRAS mutants, such as G13D, R68M and A59T, differed depending on the inhibitor. The study also demonstrated that combined treatment with the SOS1 inhibitor BI-3406 plus the MEK inhibitor trametinib effectively reduced the viability of KRAS G12C-mutant NSCLC cells, in which the Y96D or Y96S mutant was exogenously introduced. Therefore, in addition to the genetic alterations that reactivate RAS pathways, KRAS Y96C/Y96D/Y96S mutations that hamper the interaction with covalent KRAS G12C inhibitors are likely critical for acquired resistance. The resistance mechanisms described above are summarized in KEAP1 mutations were less responsive to sotorasib, irrespective of their LKB1 mutation status: the response rates for LKB1-mutant/KEAP1-mutant and wild-type LKB1/KEAP1-mutant patients were 23% (3/13) and 14% (1/7), respectively [KEAP1 mutations.Recently, several studies have revealed that secondary in NSCLC . KRAS mudagrasib . In thisotherapy . In thatdagrasib . Both Y9KRAS-mutated lung cancer cells [KRAS G12C-carrying tumors were shown to express higher levels of genes relevant to EMT [KRAS-mutated NSCLC cells, and a combination treatment with sotorasib plus the SHP2 inhibitor SHP099 and the PI3K inhibitor GDC-0941 effectively impaired the growth of KRAS G12C-mutant tumors [An epithelial-to-mesenchymal cell transition (EMT) program contributes to the therapeutic resistance to various types of anticancer drugs, including EGFR-TKIs ,86,87,88er cells . Additiot to EMT . A recent tumors .KRAS G12C or KRAS G12V significantly but incompletely suppressed tumor growth in four NSCLC cell lines, all of which retained the wild-type KRAS allele [KRAS-mutated NSCLC cells to p38 and EGFR inhibitors to varying degrees. We also reported that dual p38 and MEK inhibition effectively impaired the tumor growth of KRAS-mutated NSCLC cells [KRAS-mutated colorectal cancer cells [KRAS-mutated LUAD cells [KRAS-mutated NSCLC tumors in experimental models [KRAS-mutant NSCLC cells; a combination therapy with the MEK inhibitor trametinib plus the EGFR-TKI afatinib was effective for epithelial-like tumors, while the dual blockade of MEK and FGFR effectively suppressed mesenchymal tumor growth [KRAS-mutated tumors, whereas the growth-inhibitory effects of ARS-1620 plus afatinib or cetuximab varied among KRAS-mutated cell lines [KRAS-mutated tumors.Oncogenic RAS upregulates downstream pathways and negatively regulates RTK signaling, whereas attenuating oncogenic RAS results in compensatory activation of RTK and wild-type RAS signaling pathways . Thus, mS allele . In thatLC cells . Other rer cells  and KRASAD cells . These fl models ,98,99. Ir growth . A recenll lines . TogethePTPN11, in cancer [KRAS-driven murine models of NSCLC, genetic deletion of PTPN11 suppressed tumor development, while the dual inhibition of SHP2 and MEK resulted in sustained tumor growth control in murine- and human patient-derived organoids and xenograft models of NSCLC [KRAS-mutated NSCLC cells [KRAS mutations. The clinical efficacies of combinations of KRAS G12C inhibitors plus SHP2 inhibitors are currently under investigation in clinical trials. Altogether, it is likely that combined therapy, including the targeting of oncogenic KRAS, will be required for effective treatment given the vast intratumoral heterogeneity of KRAS-mutated tumors and the involvement of KRAS in numerous effector pathways that mediate tumorigenesis [KRAS-mutated NSCLC patients (Accumulating evidence has provided promising results regarding inhibitors of the nonreceptor protein tyrosine phosphatase SHP2, encoded by n cancer . SHP2 isn cancer . SHP2 aln cancer . In mutaof NSCLC . FurtherLC cells . ConsideLC cells , combinaigenesis . The ongpatients .KRAS-mutated NSCLC. Currently, many clinical trials are ongoing to evaluate the efficiencies of KRAS G12 inhibitor monotherapy and its combination with various types of anticancer drugs. Considering the vast heterogeneity of tumors driven by mutant KRAS, it is plausible that covalent KRAS G12C inhibitors will be therapeutically effective when combined with other anticancer drugs. Thus, the battle against oncogenic KRAS, moving toward improvements in the prognosis of NSCLC patients, has just begun.The discovery of covalent KRAS G12 inhibitors has produced dramatic advances in the treatment of


S1 File(TIF)Click here for additional data file.S3 FigDRhoGEF2, and (III) GMR-GAL4/UAS-mycPDZ-RhoGEF. (A) Scanning electron micrographs of adult eyes with ectopic expression of DRhoGEF2 or mycPDZ-RhoGEF under the control of GMR-GAL4. Scale bar = 200 μm. (B) Toluidine blue-stained transverse sections of the adult eye with DRhoGEF2 or PDZ-RhoGEF overexpression. (C) Acridine orange (AO) staining in the 3rd instar larval eye imaginal discs with DRhoGEF2 or mycPDZ-RhoGEF overexpression. (D) Cell proliferation in DRhoGEF2- or PDZ-RhoGEF-overexpressing 3rd instar larval eye imaginal discs, determined by BrdU incorporation.  Phosphorylation of dPKB/dAkt in the 3rd instar larval eye imaginal discs of control GMR-GAL4 flies  or flies overexpressing DRhoGEF2 (E) or PDZ-RhoGEF (F). Results shown in panels E and F were obtained in the same western blot experiment for which underlying data are in Labels in (A-D) indicate samples with the following genotypes: (I) GMR-GAL4/+, (II) GMR-GAL4/UAS-(TIF)Click here for additional data file.


The successful development of several coronavirus disease 2019 (COVID-19) vaccines has substantially reduced morbidity and mortality in regions of the world where the vaccines have been deployed. However, in the wake of the emergence of viral variants that are able to evade vaccine-induced neutralizing antibodies, real-world vaccine efficacy has begun to show differences across the two approved mRNA platforms, BNT162b2 and mRNA-1273; these findings suggest that subtle variation in immune responses induced by the BNT162b2 and mRNA-1273 vaccines may confer differential protection. Given our emerging appreciation for the importance of additional antibody functions beyond neutralization, we profiled the post-boost binding and functional capacity of humoral immune responses induced by the BNT162b2 and mRNA-1273 vaccines in a cohort of hospital staff. Both vaccines induced robust humoral immune responses to wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to variants of concern. However, differences emerged across epitope-specific responses, with higher concentrations of receptor binding domain (RBD)- and N-terminal domain-specific IgA observed in recipients of mRNA-1273. Antibodies eliciting neutrophil phagocytosis and natural killer cell activation were also increased in mRNA-1273 vaccine recipients as compared to BNT162b2 recipients. RBD-specific antibody depletion highlighted the different roles of non-RBD-specific antibody effector functions induced across the mRNA vaccines. These data provide insights into potential differences in protective immunity conferred by these vaccines. COVID-19 mRNA vaccines induce robust humoral immune responses with differences in epitope recognition and antibody-mediated functional properties. The unprecedented and rapid generation of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines marked a breakthrough in vaccine development and provided hope for an end to the coronavirus disease 2019 (COVID-19) pandemic. However, rising numbers of breakthrough infections, driven by evolving variants of concern (VOCs) in the setting of waning immunity, have clearly illustrated the urgent need to define correlates of immunity. Preliminary immune correlates analyses have shown a strong relationship between neutralizing antibody concentrations and vaccine efficacy 1. Yet aBeyond their role in binding and neutralization, antibodies mediate a wide array of additional immunological functions through their ability to recruit the immune system using Fc receptors (FcRs) and complement 7. Fc-meRobust protection was observed in phase 3 trials of Pfizer/BioNTech BNT162b2 4 and MoHere, we compared the humoral response across the BNT162b2 and mRNA-1273 at peak immunogenicity in a group of hospital workers. Both vaccines induced robust functional humoral immune responses, yet differences were noted in the vaccine-induced antibody profiles across the vaccine groups, with higher receptor binding domain (RBD)- and N-terminal domain (NTD)-specific IgA, as well as functional antibodies, among mRNA-1273 immunized vaccinees. Both mRNA vaccines drove robust responses against VOCs, including the beta and delta variants. Moreover, RBD-specific antibody depletion highlighted the presence of non-RBD-specific antibody effector function deployed by both platforms, albeit at different concentrations, providing evidence to explain the differential Fc-mediated effector functions observed.Seventy-three participants were included in this study. Twenty-eight received mRNA-1273 and 45 received BNT162b2. These samples were from a hospital-wide biorepository of vaccinated individuals who received an mRNA COVID-19 vaccine and had serum available for analysis following their second vaccine dose . Both van = 28) and BNT162b2 (n = 45) vaccines  (fig. S1) and supervised least absolute shrinkage and selection operator (LASSO) feature selection coupled to visualization with partial least squares discriminant analysis (PLSDA) provides multivariate resolution to determine whether overall antibody profiles differ across the vaccines . Interes= 0.008 and 0.022, respectively). Conversely, IgA responses were amplified in the mRNA-1273 immunized individuals for Alpha and Gamma VOCs   . LASSO-Pctively) . The proctively) . Addition = 16) and BNT162b (n = 15) vaccines and 10 convalescent individuals  and tested for antibody-mediated effector functions 13, 14Despite the remarkable protective immunity observed in BNT162b2 and mRNA-1273 vaccine studies against the original SARS-CoV-2 variant, breakthrough infections are on the rise globally among vaccinees 35. HoweAlthough no difference in neutralizing activity has been reported across the BNT162b2 and mRNA-1273 vaccines 27, diffThe majority of mutations in the VOCs occur in NTD and RBD 46, whicThe rapid spread of the Delta and Omicron VOCs has raised concerns globally about vaccine efficacy and the need for additional boosters. However, in previously vaccinated individuals, most Omicron infections do not require hospitalization 47–49.There are several limitations to our study. First, the mRNA vaccinated individuals were part of a hospital-wide vaccination clinic and were mostly healthy young female health care workers (median age 32 for both mRNA-1273 and BNT162b2 group) from a single site. This prevented the analysis of demographic, age, or co-morbidity related differences in vaccine responses. However, previous studies suggest that age and sex minimally influence the mRNA vaccine response 52. MoreIn conclusion, both mRNA vaccines induced robust functional humoral immune responses, with differences in epitope recognition and antibody-mediated functional properties. Additionally, strong responses against VOCs, including the beta and delta variants, were observed. RBD-specific antibody depletion highlighted the different roles of non-RBD-specific antibody effector functions induced across the mRNA vaccines, which might provide insights into potential differences in protective immunity conferred by these vaccines.The Beth Israel Deaconess Medical Center institutional review board approved this study (#2021P000344) and the parent biorepository study (#2020P000361); participants provided written informed consent. Hospital staff of at least 18 years of age planning to receive an mRNA COVID-19 vaccine from December 2020 through February 2021 were enrolled and samples were collected in a more extensive hospital-wide, prospective data and tissue biorepository. Participants self-referred from flyers posted in the hospital vaccine clinics. All participants provided blood samples collected close to each vaccine dose and two to eight weeks after the second dose for the mRNA-1273 or BNT162b2 vaccine. Seventy-three participants, 28 receiving mRNA-1273 and 45 receiving BNT162b2, were included here, providing over 90% power to detect significant differences, based on a 10,000 Monte Carlo simulation and two-sided Wilcoxon rank sum test with type 1 error rate of 5% using previously acquired data 16. The Industry employees (Space Exploration Technologies Corp.) were volunteer-tested for COVID-19, starting in April 2020. Participants completed a study survey including the collection of COVID-19-related symptoms. Blood samples were collected for serology quarterly. The cohort largely included mild, symptomatic infections 53. AlthAntigens used for Luminex based assays included SARS-CoV-2 D614G spike protein , SARS-CoV-2 S1 protein , SARS-CoV-2 S2 protein , and SARS-CoV-2 RBD , as well as antigens from SARS-CoV-2 VOCs, such as Alpha (B.1.1.7) spike protein (LakePharma), Beta (B.1.351) spike protein (LakePharma), Gamma P1 spike protein (LakePharma), Kappa B.1.617.1 spike protein  and Delta B.1.617.2 spike protein . Alpha (B.1.1.7), Beta (B.1.351), Gamma P1, Kappa (B.1.617.1) and Delta (B.1.617.2) RBDs were kindly provided by Florian Krammer, Icahn School of Medicine at Mount Sinai.Serum samples were analyzed by customized Luminex assay to quantify the relative concentration of antigen-specific antibody isotypes, subclasses, and FcγR binding profiles, as previously described 56, 57Antibody-dependent complement deposition (ADCD) was conducted as previously described 59. Brie5 cells/ml in R10 media (RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 5% penicillin/streptomycin , 5% L-glutamine , and 5% Hepes buffer (pH 7.2) ) at 37°C, 5% CO2. The THP-1 based assay was optimized using good clinical laboratory practice (GCLP) standards, aiming to define the role of antibodies in shaping monocyte phagocytosis. However, the assay can be easily adapted to profile macrophage-like or dendritic cell-like activity through differential THP-1 maturation protocols. The protocol was established to maximize signal to noise for unmanipulated THP-1 activity. For ADNP, granulocytes were isolated from whole blood by lysing red blood cells (RBCs) in ACK lysis buffer (1:10 blood in ACK lysis buffer) for 7 min before precipitation by centrifugation. Granulocytes were washed twice with cold phosphate-buffered saline (PBS) and resuspended at 2.5×105 cells/ml in R10 media; 50,000 cells per well were added to each well and incubated with immune complexes for 1 hour at 37°C, 5% CO2. For ADNP, RBC-lysed whole blood was washed, stained for CD66b+  BioLegend, cat # 305112) to identify neutrophils, and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of cells that had phagocytosed beads as well as the number of beads that had been phagocytosed  (fig. S3). Flow cytometry was performed with 5 Laser LSR Fortessa Flow Cytometer, and analysis was performed using FlowJo V10.7.1.ADCP and ADNP were conducted according to the previously described protocols 60–62.4 NK cells per well (200μl/well) and incubated for 5 hours at 37°C. NK cells were surface stained with anti-CD3 Pacific Blue , anti-CD16 allophycocyanin (APC)-Cy5 , and anti-CD56 PE-Cy7  in PBS . Samples were incubated in the dark at room temperature for 15 min. Cells were washed twice with PBS, permeabilized , and washed twice with PBS again. MIP-1β-PE  was diluted in Perm B buffer (1:50 dilution) and added to each well (50μl). Samples were incubated in the dark for 15 min at room temperature. Cells were washed twice with PBS and resuspended in the final volume of 35μl PBS per well. Flow cytometry was performed with an iQue flow cytometer (Intellicyt). NK cells were gated as CD3-, CD16+, CD56+ cells, and NK cell activity was determined as the percent of NK cells positive for CD107a and MIP-1β (fig. S3).Analysis of ADNKA was performed as described previously 63. BrieD614G SARS-CoV-2 RBD-coated magnetic beads (ACROBiosystems) were prepared according to the manufacturer’s protocol and resuspended in ultrapure water at 1 mg/ml. Beads were washed three times in PBS with 0.05% BSA using a magnet. Serum samples were incubated with beads at 3:1 beads:serum ratio, rotating overnight at 4°C. A magnet was used to deplete beads with surface-bound RBD-specific antibodies. A mock depletion (pre-depletion samples) was performed by adding 150 μl of PBS and 0.05% BSA and incubating rotating overnight at 4°C. A standard ELISA was performed to confirm RBD-specific antibody depletion. Functional assays were performed with pre- and post-depletion samples.t test.Data analysis was performed using R version 4.0.2 (2020-06-22). All Luminex data were log-transformed, and all features were scaled. Comparisons between vaccination arms were performed using a Mann-Whitney U-test test followed by a Benjamini Hochberg (BH) correction. Antigen responses (such as D614G to Alpha) were compared using the Wilcoxon-signed rank test followed by BH correction. For RBD-specific antibody depletion, all data were Z-scored to visualize and compare differences in pre- and post-depletion functional results, and comparisons between samples were performed using paired Prior to any multivariate analysis, all data were normalized using Z-scoring. Multivariate classification models were trained to discriminate between individuals vaccinated with BNT162b2 and individuals vaccinated with mRNA-1273 using all the measured antibody responses. A PCA model was constructed using all antibody variables, including antibody titer, FcR measurements, and effector functions. Maximum separation was achieved in the two-dimensional space of PC1 (37.8%) versus PC2 (11.6%). A LASSO-PLSDA model was built using a combination of the least absolute shrinkage and selection operator (LASSO) for feature selection and then classification using partial least square discriminant analysis (PLS-DA) with the LASSO-selected features. Models were calculated and figures were generated using R package “ropls” version 1.20.0 64 and “