Compared with traditional therapies, targeted therapy has merits in selectivity, efficacy, and tolerability. Small molecule inhibitors are one of the primary targeted therapies for cancer. Due to their advantages in a wide range of targets, convenient medication, and the ability to penetrate into the central nervous system, many efforts have been devoted to developing more small molecule inhibitors. To date, 88 small molecule inhibitors have been approved by the United States Food and Drug Administration to treat cancers. Despite remarkable progress, small molecule inhibitors in cancer treatment still face many obstacles, such as low response rate, short duration of response, toxicity, biomarkers, and resistance. To better promote the development of small molecule inhibitors targeting cancers, we comprehensively reviewed small molecule inhibitors involved in all the approved agents and pivotal drug candidates in clinical trials arranged by the signaling pathways and the classification of small molecule inhibitors. We discussed lessons learned from the development of these agents, the proper strategies to overcome resistance arising from different mechanisms, and combination therapies concerned with small molecule inhibitors. Through our review, we hoped to provide insights and perspectives for the research and development of small molecule inhibitors in cancer treatment. Small molecule inhibitors have been permitted to be used in post‐line therapy, first‐line treatment, and adjuvant therapy. Compared with post‐line and first‐line treatment, attempts at adjuvant therapy are just beginning. Several factors should be considered in the adjuvant setting, including other standard treatments, subsequent therapy, financial benefit ratio, and treatment‐related side effects. Cancer chemotherapy and radiation therapy have some selectivity for tumor cells because of their increased proliferation rate.Antibody therapies and small molecule inhibitors are the two main methods for targeted cancer treatment.The targets of these drugs cover a large scope. Most small molecule inhibitors belong to protein kinase inhibitors.In our review, we first describe the classification of small molecule inhibitors, including selective small molecule kinase inhibitors, selective small molecule nonkinase inhibitors, and multikinase small molecule inhibitors. Then, we discuss the small molecule inhibitors according to signaling pathways and the classification of small molecule inhibitors. In each part of specific inhibitors, we first introduce this target and the pathways involved in the target, as well as its significance in tumors. Followed by discussing United States Food and Drug Administration (US FDA)‐approved small molecule inhibitors. We subsequently introduce small molecule inhibitors in clinical trials summarized from clinical trials website. At the end of our review, we also discuss the main issues and future directions concerned with small molecule inhibitors.2To our knowledge, there are 88 small molecule inhibitors approved by the US FDA for oncology indications by August 2022Selective small molecule inhibitors usually bind to a single target and inhibit the target‐related cell signaling. A subset of cancers strongly relies on a few dysfunctions related to growth, survival, apoptosis, differentiation, cancer metabolism, and even immune modulation.Selective small molecule inhibitors are further divided into selective small molecule kinase inhibitors and selective small molecule nonkinase inhibitors.Both selective small molecule kinase inhibitors and multikinase small molecule inhibitors belong to protein kinase inhibitors.The highly dynamic nature of protein kinases allows for the design of inhibitors that recognize the active or multiple inactive conformations. Correspondingly, protein kinase inhibitors are classified into six types (type I–VI) based on their biochemical mechanisms of action.In our review, we will introduce them in the order of selective small molecule kinase inhibitors, selective small molecule nonkinase inhibitors, and multikinase small molecule inhibitors.3The number of selective small molecule kinase inhibitors is the largest in the US FDA‐approved small molecule inhibitors. This part will initially describe the first approved ABL kinase inhibitor, followed by receptor‐related kinase inhibitors like human epidermal growth factor recepter (HER), anaplastic lymphoma kinase (ALK), and fibroblast growth factor receptor (FGFR) kinase inhibitors. Then, attention will be given to kinase inhibitors targeted intracellular signaling pathways involving RAF/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT3 signaling. Finally, inhibitors targeting other cytoplasmic kinases will be introduced. The selective small molecule kinase inhibitors approved by the US FDA are summarized in Table 3.1Philadelphia (Ph) chromosome translocation results in a fusion between the abelson murine leukemia viral oncogene homolog 1 (ABL1) on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22, forming an aberrant BCR–ABL1 fusion gene on chromosome 22, which encodes a 210 kDa tyrosine kinase. This BCR–ABL1 protein further triggers phosphorylation of numerous substrates to activate MAPK, PI3K/AKT/mTOR, and JAK/STAT pathways, thereby driving the uncontrolled proliferation of leukemia cells. Nearly all of patients with chronic myeloid leukemia (CML) and 20–30% of patients with acute lymphoblastic leukemia (ALL) have BCR–ABL1 gene fusions, which are the driving molecular abnormality of these diseases. Correspondingly, BCR–ABL1 fusion tyrosine kinase is a crucial target in treatment of certain leukemias.p < 0.001). The rates of complete cytogenetic response (CCyR) were 76.2%  and 14.5% , respectively (p < 0.001).Imatinib, initially approved for treating BCR–ABL1‐driven CML by the US FDA in 2001, opens a new era in using protein kinase inhibitors in cancer treatment.Three ABL1 kinase inhibitors  are granted US FDA approval for Ph‐positive ALL. Though the efficacy of single‐agent tyrosine kinase inhibitor (TKI) therapy for Ph‐positive ALL is poor, the combination of ABL1 inhibitors with conventional chemotherapy largely improves the response and survival and now is standard of care for Ph‐positive ALL.Researches on ABL1 kinase inhibitors are ongoing, mainly focused on three directions. First, new ABL1 kinase inhibitors still arouse great interest. Both flumatinib and radotinib belong to second‐generation ABL1 kinase inhibitors and have been approved for Ph‐positive CML‐CP in China and Korea, respectively. In addition, both of them have been proved to be superior to imatinib in first‐line therapy for newly diagnosed CML‐CP.3.2Stem cell factor, also known as c‐KIT ligand, exerts its biological functions by binding to and activating the receptor tyrosine kinase (RTK) c‐KIT, also known as stem cell growth factor receptor. Dimerization of c‐KIT leads phosphorylation and activation of downstream kinases in PI3K/AKT/mTOR, MAPK, and JAK/STAT pathways and further maintains cell survival, migration, and proliferation.About 99% of GISTs have an identifiable driver alteration. The KIT  and PDGFRA (mainly in exon 18 and less frequently in exon 12 or 14) mutations are the two major molecular subtypes that disrupt autoinhibitory regions of the RTK and thereby result in ligand‐independent activation, occurring in around 70 and 15% of all GISTs, respectively. GISTs without KIT and PDGFRA mutations can be divided into SDH‐deficient and SDH‐competent GISTs.p < 0.0001) and overall survival (OS) (p = 0.0034) than those whose tumors expressed exon 9 KIT mutations.p = 0.007), median time to tumor progression , and median OS .p < 0.0001).p < 0.001), significantly prolonged 5‐year OS , compared with 1‐year imatinib.p < 0.001).Before 2000, no effective therapies were available for patients with advanced GISTs, while imatinib revolutionized the treatment of patients with this disease.p = 0.05), a median progression‐free survival (PFS) of 6.3 months versus 1.0 months  and median OS of 15.1 months versus 6.6 months  in the phase III INVICTUS trial.The main imatinib‐resistance mechanisms are activating other signaling pathways and secondary KIT mutations. Second‐line sunitinib and third‐line regorafenib are both multikinase inhibitors and will be discussed below.Currently, there are still many researches on KIT kinase inhibitors. Like imatinib, other ABL1 kinase inhibitors also inhibit c‐KIT, such as dasatinib, nilotinib, and ponatinib. In a single‐arm phase II trial, ponatinib demonstrated activity in advanced GISTs with failure of prior TKI, particularly in subtype with KIT exon 11 mutations.3.3PDGFRs (PDGFRA and PDGFRB), activated in PDGF‐dependent or PDGF‐independent modes, phosphorylate substrates and engage in signaling cascades that drive physiological or pathological functions.As mentioned above, 15% of all GISTs occur PDGFRA mutations. Imatinib‐sensitive PDGFRA mutants include mutations in exon 12, 14, or indels in exon 18. GISTs with PDGFRA exon 18 D842V variant leads to primary resistance to imatinib and other type II PDGFRA/KIT TKIs and predicts poor outcomes for patients with GISTs. The D842V mutation, the most common PDGFRA mutation , became a key target for rational drug design.A large number of multikinase TKIs with inhibitory activity toward PDGFR are used to target stromal PDGFR expression, including sorafenib for hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), and radioiodine‐refractory DTC and pazopanib for RCC and soft tissue sarcoma (STS). However, tumors often cannot be controlled through single inhibition of PDGFR expression in the stroma, which is proved by two PDGFRA antibody, olaratumab and MEDI‐575. Olaratumab, combined with doxorubicin, is approved for the treatment of adult patients with STS based on a phase II trial, in which the researchers found olaratumab plus doxorubicin improved PFS and OS compared with monotherapy with doxorubicin.Some PDGFR kinase inhibitors are undergoing clinical trials. Crenolanib is a highly specific PDGFR kinase inhibitor and is proved to be safe in several phase I clinical trials.3.4The EGFR or HER family members comprise four structurally related RTKs that are EGFR/HER1, HER2, HER3, and HER4.p < 0.001) and ORR from 30.7% to 73.7% (p < 0.001).p < 0.0001), which was 9.7 months and 5.2 months , respectively, in European patients.p = 0.038).From 2003, the treatment for NSCLC progressed significantly. First‐generation EGFR TKIs, gefitinib and erlotinib, were originally designed for patients with overexpression of wild‐type EGFR.p = 0.001), ORR from 19.1 to 50.4%.p < 0.0001).p = 0.12) and median PFS  of afatinib were slightly better than those of erlotinib.p < 0.0001) and median OS , was also approved for first‐line treatment for NSCLC with EGFR exon 19 deletion or exon 21 (L858R) mutations.Second‐generation EGFR TKIs, afatinib and dacomitinib, bind irreversibly to the ATP pocket of EGFR TK, developed to circumvent drug resistance or increase efficacy.p < 0.0001) in first‐line therapy. In FLAURA, osimeritinib also showed its advantage in controlling central nervous system (CNS) lesions over erlotinib or gefitinib.p = 0.046).p < 0.001), ORR from 29 to 65%. Because of its merits, it has been approved for first‐line treatment for NSCLC with EGFR exon 19 deletion or exon 21 (L858R) mutations and EGFR T790M mutation‐positive NSCLC in 2018 and 2015, respectively. Osimertinib has also made great strides in adjuvant therapy and has already been allowed for adjuvant therapy for NSCLC with EGFR exon 19 deletion or exon 21 (L858R) mutations in 2020. The phase III ADAURA trial showed that median disease‐free survival (DFS) was not reached and 19.6 months in placebo . However, the OS data were immature.Osimertinib, third‐generation EGFR‐TKI, binds irreversibly to the activating and T790M mutation at approximately ninefold lower concentrations than wild‐type EGFR. Surprisingly, osimertinib can distribute to the brain at plasma AUC ratios of approximately 2 following oral dosing.EGFR exon 20 insertion is another observable mutation type and associated with a poor prognosis, accounting for about 1–12% of EGFR mutations.p < 0.001).p = 0.0059) and time to intervention for CNS disease compared with lapatinib plus capecitabine.p < 0.001) and OS  in patients with HER2‐positive metastatic breast cancer but also resulted in better PFS  in those with brain metastases.HER2‐targeted therapy is important for patients with HER2‐positive advanced breast cancer. First‐line treatment with the combination of trastuzumab and pertuzumab plus taxane and second‐line treatment with trastuzumab deruxtecan significantly improve the outcome of patients with advanced or metastatic HER2‐positive breast cancer, leading to great enthusiasm to develop novel anti‐HER2 agents. HER kinase inhibitors for breast cancer are primarily approved in patients whose disease refractory to traditional HER2‐directed therapies. The addition of lapatinib to capecitabine after trastuzumab plus chemotherapy in patients with HER2‐positive breast cancer has advantages in the time to progression (TTP), from 4.4 months in the placebo plus capecitabine group to 8.4 months in the lapatinib plus capecitabine group  mutations. The third‐generation EGFR TKI osimertinib is demonstrated to be superior to the first‐ and second‐generation EGFR TKIs and used in EGFR T790M mutation‐positive NSCLC and first‐line treatment and adjuvant therapy for NSCLC with EGFR exon 19 deletion or exon 21 (L858R) mutations.3.5ALK is a RTK within the insulin receptor family, comprised of an extracellular region, a single transmembrane domain, and an intracellular kinase region. ALK fusion proteins interact with a complex network of proteins and thereby drive aberrant proliferation and survival through PI3K/AKT/mTOR, MAPK, and JAK/STAT pathways. ROS proto‐oncogene 1 (ROS1) is a RTK with an unknown physiological role in humans, while ROS1 fusion proteins drive occurrence of various tumors, including glioblastomas, NSCLC, and IMTs. Though ALK and ROS1 fusion proteins existed in multiple tumor types, ALK and ROS1 kinases inhibitors are only approved for ALK or ROS1‐positive NSCLC before July 14, 2022. ALK and ROS1 rearrangements occur in 3–8% and 0.9‐2.6% of the overall NSCLC population, mostly on adenocarcinomas, with higher prevalence in nonsmokers, younger age and early‐stage brain metastasis.p < 0.001) and upgraded ORR from 45% to 74% in first‐line treatment, median PFS from 3.0 months to 7.7 months  and ORR from 20 to 65% in second‐line treatment.The first ALK/ROS1 inhibitor, crizotinib, was initially approved in 2011. The PROFILE 1014 and the PROFILE 1007 trials confirmed a survival superiority of crizotinib over platinum‐based chemotherapy in first‐line and second‐line therapy (after 1 platinum‐based chemotherapy regimen) for ALK‐positive NSCLC, respectively.p < 0.00001) versus chemotherapy.p < 0.0001).Second‐ and third‐generation ALK inhibitors are not just for post‐line therapy. Continuous researches give more choice for patients with ALK‐positive NSCLC in first‐line treatment. The first second‐generation ALK inhibitor ceritinib showed a statistically significant and clinically meaningful improvement in PFS  are a kind of rare mesenchymal tumor consisting of a variable mixture of myofibroblasts and inflammatory infiltrates that can occur throughout the body, mainly in the mesentery, retroperitoneum, and pelvis. ALK rearrangements occur in ≥50% of IMTs. On July 14, 2022, the US FDA approved crizotinib for patients with unresectable, recurrent, or refractory ALK‐positive IMTs based on a phase Ib trial (NCT01121588). The ORR was 67% for ALK‐positive IMTs with a consistent safety profile, the efficacy of which was confirmed by a phase II study (NCT01524926).Researches are ongoing for developing new ALK/ROS1 inhibitors. Ensartinib has been approved in China for ALK‐positive NSLCL. A phase III trial (NCT02767804) showed that first‐line treatment with ensartinib for ALK‐positive NSCLC had superior efficacy to crizotinib in both systemic and intracranial disease.3.6Mesenchymal–epithelial transition factor (MET), also known as the tyrosine receptor of HGF, is a single‐pass transmembrane receptor. MET homodimerization activates the MAPK and PI3K/AKT/mTOR pathways, which promote cell migration, proliferation, and survival.Two small molecule kinase inhibitors targeting MET, capmatinib and tepotinib, got accelerated approval for NSCLC with MET exon 14 skipping in 2020 and 2021, respectively. For capmatinib, the phase II GEOMETRY mono‐1 trial demonstrated that the ORR in the treatment‐naïve population was 68% and DOR was 12.6 months, with 41% and 9.7 months, respectively, in the previously treated group. At the same time, low‐grade peripheral edema and nausea were the main toxic effects.Researches on MET inhibitors are focused in three directions. First, selective MET inhibitors are explored in other types of MET‐dysregulated NSCLC and different kinds of tumors. MET amplification or overexpression attributes to one of the predominant EGFR‐TKI resistance mechanisms in patients with EGFR‐mutated NSCLC.3.7Rearranged during transfection (RET) is a transmembrane RTK whose homodimerization activates several downstream pathways, including MAPK, PI3K/AKT/mTOR, JAK/STAT, PKA, and PKC pathways, essential for the normal development and maturation of diverse tissues.Multikinase inhibitors with anti‐RET activity, such as cabozantinib and vandetanib, were initially tested in patients with advanced RET‐rearranged NSCLC.Many MET inhibitors are undergoing clinical trials. RXDX‐105 is a multikinase RET inhibitor. In phase I/Ib trial for RET inhibitor‐naïve patients with RET fusion‐positive NSCLCs, RXDX‐105 showed different ORRs between KIF5B‐RET‐containing and non‐KIF5B‐RET‐containing tumors. The ORRs were 0 and 67% for these two gene alterations, respectively (NCT01877811). The reason contributing to the differential responses is not clear.3.8Fibroblast growth factor (FGF) receptors  are RTKs consisting of three extracellular immunoglobulin (Ig)‐like domains , a transmembrane domain and two intracellular tyrosine kinase domains (TK1 and TK2).To date, three selective FGFR kinase inhibitors, erdafitinib, pemigatinib, and infigratinib, are approved for treating urothelial carcinoma or cholangiocarcinoma.These selective FGFR inhibitors have been explored in other tumors. Preclinical trials showed that erdafitinib inhibited the growth of glioma cells with FGFR3‐TACC3 fusions in vitro and in vivo. Erdafitinib manifested clinical improvement with stable disease and minor response for glioblastoma patients with FGFR3‐TACC3 rearrangements.There are many selective FGFR inhibitors at different stages of clinical trials. FGFR inhibitors with clinical activity are classified into FGFR1‐4, FGFR1‐3, and FGFR4 inhibitors. Futibatinib, rogaratinib, LY2874455, and ASP5878 belong to FGFR1‐4 inhibitors. Futibatinib is an irreversible FGFR1‐4 inhibitor. Phase I trial (NCT02052778) of futibatinib recruited patients with advanced solid tumors harboring FGF/FGFR aberrations. This trial highlighted that futibatinib was also effective in patients whose disease was already resistant to prior FGFR inhibitors.3.9NTRK1, NTRK2, and NTR3 genes encode tropomyosin receptor kinase A, B, and C , which are the members of the cell surface RTK family, regulating cell proliferation, differentiation, and apoptosis through activate pathways of PLCγ, MAPK, PI3K/AKT/mTOR, and PKC.Two agents, larotrectinib and entrectinib, have received US FDA approval for NTRK‐gene fusion locally advanced or metastatic tumors. A pooled analysis of three phase I/II clinical trials  showed that the ORR of larotrectinib was 79% in patients with TRK fusion‐positive solid tumors, with 16% of patients having CR.Similarly, developing new indications for existing drugs and exploiting new drugs are two important research directions. Trials have been done or are ongoing to find new usage scenarios. A pediatric phase I trial of neoadjuvant larotrectinib presented a promising medication for children with newly diagnosed and locally advanced TRK fusion sarcomas, which might expand the use of TRK inhibitors in neoadjuvant therapy.3.10FMS‐like tyrosine kinase 3 (FLT3), expressed in various lymphohematopoietic cells and tissues, belongs to the class III RTK family and plays an important role in regulating cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells through activating PI3K/AKT/ mTOR, MAPK, and JAK/STAT downstream signalings.p = 0.002) and OS from 25.6 months to 74.7 months . The rate of severe adverse events was similar in the two groups.p < 0.001). The percentages of patients with CR (21.1 vs. 10.5%) and CR with full or partial hematologic recovery (34.0 vs. 15.3%) were higher in the gilteritinib than in the chemotherapy group. In addition, grade 3 or higher and serious adverse events occurred less frequently in the gilteritinib group than in the chemotherapy group.Correspondingly, FLT3 inhibitors are developed to treat FLT3‐mutated AML.Researches on midostaurin and gilteritinib are ongoing. Only midostaurin has been approved for first‐line treatment of FLT3 mutant AML. A phase II study (NCT03836209) is recruiting untreated patients with FLT3 mutant AML to compare the effectiveness of gilteritinib to midostaurin in first‐line therapy. Enhancing efficacy through combination therapy is another trend. A phase I/II trial for patients with AML and MDS showed that the combination of midostaurin and a DMNT inhibitor 5‐azacytidine was an effective and safe regime, especially for patients with FLT3 mutations but not previously exposed to other FLT3 inhibitors.p = 0.02).New FLT3 inhibitors are under different research stages. The second‐generation FLT3 inhibitor quizartinib is widely studied and is the most promising drug for approval. FLT3 inhibitors are classified as either type I or II based on their interaction with their kinase targets. Type I FLT3 inhibitors target either FLT3‐ITD or FLT3‐TKD point mutations, while type II inhibitors commonly only target FLT3‐ITD.3.11The TME is rich in cytokines and their receptors, which exert complex functions in antitumor response or tumor‐promoting effect.p < 0.0001). Serious adverse events occurred in 13% of patients in the pexidartinib group, including increasing levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase and hypertension. In the ENLIVEN trial, hepatotoxicity was a dose limiting toxicity.Tenosynovial giant cell tumor (TGCT) is a rare and locally aggressive neoplasm with overexpression of CSF1. Before 2019, surgery was standard with no approved systemic therapy. The CSF1R kinase inhibitor, pexidartinib, is indicated for patients with TGCT based on the data from the phase III ENLIVEN trial in 2019, which pioneers the way for harnessing cytokines and chemokines for cancer therapy.Pexidartinib has also been explored in other tumor types. Trials of pexidartinib in advanced castration‐resistant prostate cancer, recurrent glioblastoma, and relapsed or refractory Hodgkin lymphoma have been finished, and the results are waiting to be published . A similar situation is for the phase I/II trial (NCT02452424) of pexidartinib plus anti‐PD‐1 antibody pembrolizumab for advanced melanoma and some other solid tumors.The development of novel CSF1R kinase inhibitors is relatively slow and a few monoclonal antibodies against CSF1R are also exploited to target the CSF1/CSF1R axis. Edicotinib is a selective inhibitor of the CSF1R tyrosine kinase. Phase I/II study of this agent for relapsed/refractory classical Hodgkin lymphoma demonstrated its safety profile but limited antitumor activity in treating classical Hodgkin lymphoma.3.12The MAPK cascade, also known as the RAS/RAF/MEK/ERK signaling pathway, regulates cell proliferation, differentiation, and survival.BRAF, MEK, and ERK are oncogenes encoding serine‐threonine protein kinases. Some mutations in the BRAF gene result in constitutively activated BRAF proteins and then triggering downstream MEK and ERK.p < 0.0001) and median OS from 17.4 months to 22.3 months  and the safety profile for cobimetinib plus vemurafenib was tolerable and manageable in the phase III coBRIM trial.p < 0.0001), median OS from 16.9 months to 33.6 months .Vertical blockade of BRAF and MEK in the MAPK pathway reduces paradoxical downstream activations and efficiently targets the MAPK pathway.As mentioned above, other types of cancer may harbor BRAF mutations or MAPK pathway aberrations. Correspondingly, monotherapy with BRAF or MEK inhibitors and BRAF plus MEK inhibitors also have indications in other types of tumors. Vemurafenib is indicated for the treatment of patients with BRAF V600 mutant erdheim‐chester disease.Though having not been permitted, many clinical trials have proven efficacy of BRAF or MEK inhibitors or BRAF plus MEK inhibitors in more cancer types. Phase II clinical trials showed that vemurafenib represented a potential new treatment option for BRAF‐V600E‐positive papillary thyroid cancer and NSCLC with BRAF V600 mutations.However, BRAF or MEK inhibitors are not suitable for all BRAF‐mutant cancers. Phase II clinical trials showed that single‐agent vemurafenib did not show meaningful clinical activity in patients with BRAF V600E mutant‐CRC, which may attribute to tumor specificity and feedback mechanisms and may be conquered by combination strategies.p = 0.025) without obviously adding adverse events.The clinical benefit of combining PD‐1/PD‐L1 antibody with RAF and MEK inhibitors is inconsistent. Preclinical trials provide a rationale for these combining strategies.Novel kinase inhibitors involved in RAF, MEK, and ERK are under development. New RAF inhibitors are developed to resolve more molecular alterations in RAS‐RAF‐MEK signaling. Belvarafenib is a type II RAF inhibitor and is effective in BRAF V600E‐ and NRAS‐mutant melanoma.3.13The PI3K/AKT/mTOR signaling pathway triggered by various extracellular stimuli regulates cell proliferation, survival, and angiogenesis. PI3Ks are the family of vital lipid kinases widely distributed in mammalian cells that phosphorylate PIP2 into PIP3, which activates PDK1 and then influences AKT. Activation of PI3K and AKT leads to mTOR activation and phosphorylation of S6K1 and 4e‐BP1.p < 0.001; ORR: 35.7 vs. 16.2%).p = 0.1343).p < 0.0001) (NCT01569295). In contrast, the accompanying serious adverse events and infections may prevent its approval. Compared with the PI3Kδ specific inhibitor idelalisib, the PI3Kδ/γ dual duvelisib shows a more potent inhibitory activity of PI3K protein and the US FDA has approved duvelisib for CLL or SLL after at least two prior therapies.It is reported that PIK3CA mutations occur in 40% of HR‐positive, HER2‐negative breast cancer.Though several PI3K inhibitors have been approved, efforts never stop to exploit more suitable inhibitors. Buparlisib and pictilisib belong to pan‐PI3K kinase inhibitors, which have been tested as a single agent or combination regimes in many tumor types, such as breast cancer, squamous cell carcinoma of the head and neck, glioblastoma, and lymphoma.To date, no AKT inhibitors have been approved. Several AKT inhibitors are in different clinical stages for various tumor types, especially for breast cancer, prostate cancer, and RCC, such as ipatasertib, capivasertib, afuresertib, and MK‐2206.The approved mTOR inhibitors only inhibit TORC1, which could inversely activate AKT. mTOR kinase inhibitors can inhibit TORC1 and TORC2 and are supposed to avoid resistance caused by AKT phosphorylation.3.14The Janus kinase (JAK)/signal transducer and activators of transcription (STAT) pathway plays essential roles in tumorigenesis and immune function by mediating the cellular response to cytokines, interferons, and growth factors.p < 0.001).Myelofibrosis is a kind of hematological malignancy that includes primary myelofibrosis, postpolycythaemia vera myelofibrosis, and postessential thrombocytosis myelofibrosis, and ectopically activates the JAK/STAT pathway.Several JAK inhibitors have been explored, but some of them discontinued further research due to serious adverse events or limited efficacy, such as JAK1/2 inhibitor AZD1480 and momelotinib. AZD1480 ceased in the phase I study because of unusual dose‐limiting toxicities and the lack of clinical activity.3.15Cell cycle is crucial for cell proliferation process and mainly mediated by cyclin‐dependent kinases (CDKs), cyclins, and CDK inhibitors. CDKs are the cell cycle activators and require cyclin proteins for phosphorylation of the key substrates. CDK inhibitors negatively regulates the activity of CDKs. In the human cell, at least 20 CDKs and 29 cyclins have been identified.To date, the CDK4/6 inhibitors have been proved to interfere with the proliferation of breast cancer cells by decreasing pRb phosphorylation and arresting the cell cycle in the G1 phase.In addition to the approved indications, these CDK4/6 inhibitors are also trying to broaden their use scopes. The phase II PALLET trial evaluated adding palbociclib to letrozole as neoadjuvant therapy in patients with ER‐positive early breast cancer. However, this regime did not increase the clinical response rate over 14 weeks.New CDK4/6 inhibitors are still in development. Dalpiciclib is also a CDK4/6 inhibitor and has been approved in combination with fulvestrant for HR‐positive and HER2‐negative breast cancer progressing on endocrine therapy in China.3.16Bruton's agammaglobulinemia tyrosine kinase (BTK) belongs to the Tec non‐RTK family, which is one of the largest kinase families in mammals and includes BTK, ITK, TEC, BMX, and TXK.In 2013, a first‐generation BTK inhibitor, ibrutinib, was first approved by the US FDA for mantle cell lymphoma (MCL), CLL/SLL with or without 17p deletion, waldenström's macroglobulinemia (WM), and MZL.Tirabrutinib and orelabrutinib also belong to second‐generation BTK inhibitors. Tirabrutinib binds to BTK with an IC50 of 2.2nM. The data from phase II trials indicated favorable efficacy of tirabrutinib in patients with WM and primary CNS lymphoma.In contrast to the covalent inhibitors, the third‐generation BTK inhibitors, pirtobrutinib and nemtabrutinib, noncovalently and reversibly bind to BTK kinase, do not rely on Cys481, and may further overcome the resistance caused by previous BTK inhibitors.3.17Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are important metabolic enzymes that catalyze isocitrate to α‐ketoglutarate (αKG).To date, two IDH inhibitors have been approved for AML and/or cholangiocarcinoma.p = 0.002) and OS . CR was 47% in the ivosidenib plus azacitidine arm and 15% in the placebo plus azacitidine arm. The other indication of ivosidenib is for the treatment of locally advanced or metastatic, chemotherapy refractory cholangiocarcinoma with an IDH1 mutation based on data from the phase III study AG120‐C‐005 (ClarIDHy). Ivosidenib at a dose of 500mg daily significantly improved median PFS  and resulted in a favorable OS benefit  compared with placebo.Enasidenib is an IDH2 inhibitor approved by the US FDA for relapsed or refractory AML with IDH2 mutation in 2017. At 100 mg daily dose of enasidenib, 19% of patients achieved CR and CR with partial hematological recovery (CRh) in another 4% of patients in a phase I/II study (NCT01915498).Research on drug sensitivity and resistance found several factors were associated with the IDH inhibitors. First, cooccurring RAS pathway mutations decreased the possibility of responding to ivosidenib monotherapy. Interestingly, patients with a JAK2 mutation achieved a higher percentage of a CR or CRh.Additional small molecule IDH inhibitors are under various preclinical and clinical development stages. IDH‐305, BAY‐1436032, and DS‐1001b belong to IDH1 inhibitors. In the phase I trial (NCT02381886) for IDH1‐R132‐mutant AML and MDS, IDH‐305 exhibited a narrow therapeutic window, and the following trials were hindered.3.18Nine structurally similar nonreceptor protein TKs  constitute the Src family kinases, which interact with multiple tyrosine kinase, growth factor, integrin, and G‐protein‐coupled receptors and participate in pathways promoting cell proliferation and survival.The selective Src inhibitor was not approved until 2 years ago for patients with actinic keratosis, a precancerous lesion of invasive cutaneous squamous cell carcinoma (iCSCC). Actinic keratosis may progress to iCSCC at a rate of 0.025–16% per lesion per year. Because of its unpredictable nature of progression, treatment is recommended. Cryosurgery is a common method for individual actinic keratosis lesions. Photodynamic therapy and topical agents  are recommended for multiple lesions and surrounding solar‐damaged skin. However, local reactions or long‐term medication may reduce adherence and undermine treatment success. Hence, there is a need to find other methods for actinic keratosis. In 2020, tirbanibulin, a Src‐specific kinase and tubulin polymerization inhibitor, was approved for the topical treatment of actinic keratosis based on the data from two phase III trials.Combined with other agents to increase other agents’ efficacy or avoid resistance caused by the principal agents, maybe a future for selective Src inhibitors and some studies have shown the possibilities in preclinical models. Saracatinib is a selective Src inhibitor, and it was well tolerated in patients with advanced solid malignancies in the phase I trial. In the follow‐up single‐drug studies, no good results were obtained.Selective small molecule kinase inhibitors are supposed to inhibit a single protein kinase, but they usually bind to a few structurally related kinases.4Selective small molecule nonkinase inhibitors bind to targets beyond the kinome, thereby blocking subsequent functions to control tumors.4.1Hypoxia‐inducible factor 1α (HIF‐1α) and HIF‐2α trigger HIF‐1 transcription to promote induction of various proangiogenic genes, such as VEGF, VEGFR, EGF, ANGPT, Tie‐2, TIMP‐1, and PAI‐1, and thereby play critical roles in tumor proliferation, apoptosis, metabolism, and metastasis in response to low‐oxygen concentrations in the TME, which can be hydrolysis by VHL protein.Four strategies can be used for the interference of HIF‐1 accumulation. Direct inhibition of HIF‐1 transcription by HIF‐1α or HIF‐2α inhibitor is an important method. Blocking HIF‐1 translation or signaling pathways associated with HIF‐1 collection and enhancing HIF‐1 degradation can also circumvent the roles of HIF‐1.Clinical studies of belzutifan (as monotherapy or combination therapy) for particular tumor types are ongoing, especially for RCC and pNET. Among them, 12 out of all 18 trials are for RCC. A phase III study (NCT04195750) is ready to recruit 736 participants with RCC to compare belzutifan with everolimus. Combination of belzutifan with PD‐1 inhibitor and/or multikinase inhibitor (lenvatinib or cabozantinib) is a trend to increase efficacy and avoid resistance and is under estimation in several phase II or III trials .The factors affecting the above four aspects of HIF‐1 may impact HIF‐1 accumulation. There are also HIF‐1α inhibitors under study, such as IDF‐11774 and PX‐478. IDF‐11774 is a HIF‐1α inhibitor that suppresses HSP70 chaperone activity to disrupt HIF‐1α refolding and stimulate HIF‐1α degradation. It showed antitumor activity on melanoma, prostate cancer, and thyroid cancer in vitro and/or in vivo.4.2PARP contributes to DNA damage repair (DDR) and the maintenance of genomic stability, which allow cancer cells to develop resistance to radiation and DNA damaging chemotherapeutics.Three PARP inhibitors  for ovarian cancer can be used as a single agent during maintenance therapy after response to first‐line platinum‐based chemotherapy or as monotherapy in the post‐line treatment.In breast cancer, olaparib and talazoparib demonstrated significant clinical benefits over chemotherapy in PFS and ORR for germline BRCA‐mutated, HER2‐negative metastatic or locally advanced breast cancer based on the phase III OlympiAD and EMBRACA trials, respectively. Notably, grade ≥3 adverse events in the olaparib arm were fewer than in the chemotherapy arm (36.6 vs. 50.5%). Compared with chemotherapy, talazoparib brought a 17% increase in grade 3–4 hematologic adverse events and a 6% decrease in the rate of nonhematologic grade 3 events.p < 0.001) and OS  compared with enzalutamide or abiraterone. Anemia and nausea were the main toxic effects in patients who received olaparib.In addition, two PARP inhibitors, olaparib and rucaparib, received US FDA designation for metastatic castration‐resistant prostate cancer with homologous recombination repair gene mutation or BRCA mutation or ATM mutation.The approved PARP inhibitors are developing in two main directions. First, the therapeutic scope of PARP inhibitors is expanding to other tumor types, most with BRCA or ATM mutations, such as endometrial, urothelial, SCLC, STS, mesothelioma, and gastric cancer.New PARP inhibitors are ongoing in different clinical stages, such as, pamiparib, fluzoparib, iniparib, veliparib, and stenoparib.Two other questions about PARP inhibitors are also important. First, the factors associated with the sensitivity of PARP inhibitors are studied extensively. As mentioned above, patients whose tumors harbor BRCA or ATM or other mutations or HRD are likely to respond to PARP inhibition. An HRD phenotype is associated with genomic scars and mutational signatures, which can be used for defining broader populations that may benefit from DDR‐targeted drugs. Moreover, there is a strong association between HRD and ovarian cancer platinum sensitivity. Therefore, platinum sensitivity functions as a surrogate marker for HRD.4.3Tumor cells are not only activated by genetic alterations but also often use epigenetic processes to ensure their survival despite of various interference. Since epigenetic pathways exhibit greater flexibility of several orders of magnitude relative to genetic alterations, epigenetic alterations are receiving increasing attention.4.3.1DNA methylation is one of the best‐described epigenetic events that controls gene expression and is dysregulated in diseases like cancer.p < 0.001), delayed time to transformation to AML or death , and improved quality of life compared with supportive care.p < 0.001) and RFS  compared with placebo.p = 0.005) compared with conventional care regimes.Azacytidine is the first approved DNMT inhibitor for MDS, AML, and juvenile myelomonocytic leukemia (JMML). The phase III cancer and leukemia group B trial for higher‐risk MDS showed azacytidine treatment enhanced treatment response . Moreover, a phase II study (ChiCTR‐IIR‐16008182) found that rhG‐CSF combined with minimal‐dose decitabine maintenance after allo‐HSCT can reduce the incidence of relapse in patients with high‐risk AML.Similarly, decitabine was licensed for MDS based on several studies demonstrating an ORR of 49% in elderly patients with high‐risk MDS, reduction of AML transformation, and improvements in patient‐reported quality of life.p = 0.003).Several aspects concerned with DNMT inhibitors deserve attention, including toxicity, efficacy, and combination therapy. First, the most common grade 3–4 adverse event was cytopenia, including neutropenia and thrombocytopenia. Clinically, there are corresponding drugs for myelosuppression, so it is not difficult to address. Second, several factors may affect the efficacy of DNMT inhibitors. Unfavorable‐risk cytogenetic abnormalities and TP53 mutations were correlated with favorable clinical response and robust (but incomplete) mutation clearance in AML and MDS treated by decitabine.Many other DNMT inhibitors are under preclinical or different clinical stages, such as guadecitabine, zebularine, and CP‐4200. The latter two drugs are in the preclinical phase.4.3.2Proteins of the histone family are conserved alkaline proteins in eukaryotes and play pivotal roles in DNA packaging and gene regulation. Histone acetylation is associated with active transcription and is mainly localized at enhancer regions, promoters, and the gene body.p < 0.0001) when treating patients with relapsed or refractory multiple myeloma.To date, four HDAC inhibitors  have been approved to treat hematological malignancies.p = 0.86).p = 0.01).131I‐metaiodobenzylguanidine for patients with relapsed or refractory neuroblastoma was 32% and the value was 14% in the 131I‐metaiodobenzylguanidine arm.HDAC inhibitors as a single agent or a part of combination regimes have been explored in other solid and hematological tumors, such as NSCLC, gastrointestinal carcinoma, malignant pleural mesothelioma, neuroblastoma, AML, MDS, and so on.Many HDAC inhibitors are underdeveloped, including, but not limited to, pracinostat, quisinostat, dacinostat, AR‐42, mocetinostat, and citarinostat. Pracinostat is a pan‐HDAC inhibitor and has been evaluated in the phase II clinical stage for various of solid and hematological tumors, including MDS, AML, prostate cancer, and sarcoma .4.4The nuclear export receptor XPO1, also known as CRM1, with a pleiotropic role in transporting a plethora of proteins and RNA species, including rRNAs, snRNAs, mRNA, microRNAs, and tRNAs, is in charge of the nuclear export of tumor suppressor protein and growth regulators from the nucleus to the cytoplasm.p = 0.0075). Grade 3–4 adverse events, including thrombocytopenia, fatigue, and anemia, were higher in the three‐drug combination.The XPO1 inhibitor selinexor covalently and reversibly interacts with Cys528 in the nuclear export signal‐binding pocket of XPO1. Therefore, it restores tumor suppressor function and eventually causes tumor cell apoptosis.p = 0.011). Besides, the absence of CALB1 predicted a longer PFS with selinexor compared with placebo . Therefore, the expression of CALB1 may be used as a biomarker to choose suitable patients with dedifferentiated liposarcoma for the treatment of selinexor.Clinical trials of selinexor for dedifferentiated liposarcoma, recurrent glioblastoma, NHL, and AML also received positive results. Among them, the studies of dedifferentiated liposarcoma and recurrent glioblastoma have advanced to clinical phase II and/or phase III.Eltanexor and verdinexor also belong to XPO1 inhibitors, the binding mode of which is similar to that of selinexor.4.5C‐X‐C‐motif chemokine receptor 4 (CXCR4) is a G‐protein‐coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems and also has important roles in promoting tumor cell proliferation, metastasis, and angiogenesis.To date, none of the CXCR4 inhibitors are approved directly to target tumors. The CXCR4 inhibitor plerixafor is approved for patients with NHL or multiple myeloma to mobilize hematopoietic stem cells into the peripheral blood for autologous transplantation.There are many other small molecule CXCR4 inhibitors for cancers in development, namely small peptide molecule and small nonpeptide molecule inhibitors.4.6The principal upstream factor of the MAPK pathway is RAS protein, which is a small, membrane‐bound guanine nucleotide‐binding GTPase and concludes four members, including HRAS, NRAS, KRAS4A, and KRAS4B. The KRAS4A and KRAS4B are two isoforms of KRAS.Once, RAS protein was thought to be untargetable because of lacking binding pockets on its protein surface.There are other RAS inhibitors or agents of other mechanisms under development for targeting RAS mutant tumors. Adagrasib is the most promising agent. It is also a selective and irreversible KRAS G12C inhibitor. In the phase II study (NCT03785249) of this agent for patients with KRAS G12C mutant NSCLC previously treated with PD‐1/PD‐L1 antibody and platinum‐based chemotherapy, the ORR reached 42.9% and the median DOR was 8.5 months. Besides, the intracranial confirmed ORR was 33.3% and the safety profile was acceptable.4.7mTOR, a classical downstream effector of the PI3K pathway, is usually assembled in two complexes, mTOR complex 1 (mTORC1) and mTORC2, with different regulatory mechanisms.p = 0.0078) and PFS  compared with interferon in previously untreated RCC. Everolimus was superior to placebo for PFS in metastatic RCC after treatment with sunitinib, sorafenib, or both.Rapamycin, also known as sirolimus, and its analogs (temsirolimus and everolimus) constitute the first generation of mTOR inhibitors, which bind to FKBP12 and preferentially inhibit mTORC1.4.8Hedgehog (Hh) signaling plays a pivotal role in embryonic development, tissue homeostasis, regeneration, and stem maintenance in adults. In the absence of Hh ligands, the transmembrane Patched receptor (PTCH) antagonize the activation of smoothened (SMO). Upon Hh ligand binding, the PTCH receptor is exported from the cilium allowing SMO to enter and to activate downstream elements of the pathway. SMO activation promotes the dissociation of glioma‐associated oncogene (GLI) transcriptional factors GLI1, GLI2, and GLI3, from their negative regulator SUFU, and subsequently induce nuclear translocation. Its deregulation in its regulators such as PTCH, SMO, GLI1, and GLI2 causes many different tumors, including basal cell carcinoma (BCC), AML, and several solid cancers.The US FDA accepted three SMO antagonists , to treat BCC or AML.While serious adverse events and drug resistance hinder their use in the clinic. Many efforts have been paid to solve these problems.The approved SMO inhibitors are being tested in other tumor types, and novel SMO inhibitors are developing. Phase I study (NCT01125800) of sonidegib in pediatric brain and solid tumors demonstrated that sonidegib was well tolerated.4.9Anticancer therapies mainly involve viral and cellular oncogenes, cellular proliferation, and transformation. However, programmed cell death was deemed a logical and realistic therapeutic strategy until the discovery of apoptosis after tumor cells exposure to glucocorticoids, cytotoxic agents, or radiation.p < 0.001).To date, only the BCL‐2 inhibitor venetoclax has been approved for treating hematological tumors. Venetoclax belongs to BH3 mimetics, which are small molecules that mimic the binding of BH3‐only proteins to antiapoptotic BCL‐2 proteins and therefore block antiapoptotic function. BH3‐only proteins are a subclass of proapoptotic BCL‐2 proteins that contain only one BCL‐2 homology domain, which can bind to the groove formed by four BCL‐2 homology domains of antiapoptotic members of the BCL‐2 family. The BCL‐2 inhibitors are designed to imitate the structure of BH3‐only proteins.t  translocation. Phase I study (NCT03314181) combining venetoclax with CD38 antibody daratumumab and dexamethasone demonstrated a high rate of deep and durable responses in patients with relapsed or refractory multiple myeloma, especially for patients with relapsed or refractory multiple myeloma those with t .Expanding the indications of venetoclax and achieving durable and deep response through combination regimes are two developmental directions for venetoclax. Many tumor types, especially for other hematological tumors are under investigation, such as WM, ALL, and multiple myeloma. Venetoclax was tested in patients with previously treated WM in a phase II study (NCT02677324), which found venetoclax was safe and high active, even in patients progressing on BTK inhibitors.Several resistant mechanisms to venetoclax have been described, including alterations of other BCL‐2 proteins’ expression, mutations in the BCL‐2 protein, and modifications in mitochondrial function. For example, increased expression of BCL‐XL is associated with resistance to venetoclax in MCL. Mutations in the BCL‐2 protein, such as G101V and F104L, could largely reduce the BCL‐2 binding affinity for venetoclax compared with wild‐type BCL‐2. Combinatorial strategies may conquer the resistance.Selective small molecule inhibitors are often used in patients with the presence or absence of specific predictive molecular alteration detected from tumor tissue or body fluid sampling such as blood.5As mentioned above, multikinase inhibitors have a wide range of targets. They block the VEGF/VEGFR signaling pathway while also inhibiting other pathways to exert antiangiogenic and antiproliferative effects.p = 0.03).p < 0.001).p = 0.016). Moreover, the proportion of hypertension with tivozanib (the most common grade 3 or 4 treatment‐related adverse event) was lower than with sorafenib (14 vs. 20%).Clear cell RCC is the most common type of RCC and is rich in vascular networks induced by dysregulation of the VHL/HIF pathway, which provides a scientific rationale for developing VEGFR‐focused multikinase inhibitors  in this tumor type.p < 0.001).p < 0.0001) compared with placebo.p = 0.005) and median PFS  was longer with cabozantinib than with placebo. The ORRs were 4% and less than 1%, respectively (p = 0.009).Sorafenib was the first agent approved for patients with advanced HCC based on the phase III SHARP trial, which revealed survival benefits with sorafenib versus placebo .p < 0.001) and increased response rate .p < 0.0001).p < 0.001) and ORR (p < 0.001).In thyroid cancer, sorafenib, lenvatinib, and cabozantinib have received US FDA approval to treat radioiodine‐refractory differential thyroid cancer based on phase III trials . Median PFS in the sorafenib arm was 10.8 months and the value was 5.8 months in the placebo arm  and median PFS from 6.0 weeks to 24.1 weeks .p < 0.0001) and DCR was 52.6 and 9.1%, respectively. No significant difference in OS between study arms was found, because 84.8% of patients in the control arm crossed over to regorafenib.p < 0.0001).Advanced imatinib‐resistant GISTs respond to second‐line sunitinib and third‐line regorafenib, both of which not only inhibit KIT but also PDGFR and/or VEGFR family members.Several other factors concerned with multikinase inhibitors deserve attention. First, in addition to the above diseases for multikinase inhibitors, they also have other indications: sunitinib for pNET, pazopanib for STS, regorafenib for metastatic CRC, and lenvatinib for endometrial carcinoma.Many small molecule inhibitors targeting VEGFR are still in clinical trials and can be divided into selective VEGFR kinase inhibitors, dual mechanism inhibitors, and multikinase inhibitors Table . Among tp < 0.001) and OS  compared with placebo.The development of selective VEGFR kinase inhibitors is limited. Vatalanib and semaxanib belong to selective VEGFR2 kinase inhibitors. Vatalanib mainly targets VEGFR tyrosine kinases in submicromolar range but it also inhibits PDGFR, KIT, and c‐Fms at higher concentrations. The antiangiogenic drug vatalanib added to chemotherapy with FOLFOX4 failed to show advantages in OS and PFS compared with FOLFOX4 in two phase III trials for first‐ and second‐line treatment of CRC (CONFIRM 1 and 2).p < 0.001) and OS  in patients with advanced NSCLC progressing after second‐line or further treatment compared with a matched placebo.p < 0.001) and OS  with apatinib than with a placebo.p = 0.245) and fewer adverse events  versus sorafenib.As mentioned above, multikinase inhibitors have made great success in treating cancers. In addition to the US FDA‐approved multikinase inhibitors, anlotinib, apatinib, donafenib, and surufatinib have received approval in China. Anlotinib was licensed for NSCLC and STS. The phase III ALTER 0303 trial showed that anlotinib improved PFS .6The development and approval of small molecule inhibitors have essentially transformed multiple cancers’ clinical care and improved prognosis in many tumors.6.1It is reported that at least one alteration in genes can be detected in approximately 40% of cancers.6.2The development of small molecule inhibitors is often initially used for post‐line therapy, gradually transitioning to first‐line treatment, and finally trying to be applied in adjuvant and neoadjuvant settings.To date, several agents have received the permits for patients with early malignancies after surgery, such as imatinib for KIT or PDGFRA mutant GISTS, osimertinib for NSCLC with EGFR exon 19 deletion or exon 21 (L858R) mutations, neratinib for HER2‐positive breast cancer, dabrafenib plus trametinib for melanoma with BRAF V600E or V600K mutations, and sunitinib for RCC.Though many efforts have been made for the neoadjuvant treatment of cancers with promising outcomes, no targeted drug has yet been approved. Although not yet approved, scientists have not stopped exploring. As mentioned above, numerous small molecule inhibitors have been explored in the field of neoadjuvant therapy, such as MET inhibitor capmatinib for NSCLC, TRK inhibitor larotrectinib for sarcomas, PI3K inhibitor taselisib, CDK4/6 inhibitor palbociclib, and AKT inhibitor MK‐2206 for breast cancer. 6.3It is crucial to balance the drugs’ efficacy and toxicity. Nearly all the small molecule inhibitors have a chance to be halted in more or less percent of patients because of severe side effects.6.4p < 0.0001).We should avoid abrupt discontinuation of small molecule inhibitors without any other systematic treatment when the patients encounter the onset of progression. Because of differences in growth kinetics of responsive and resistant tumor subclones, abrupt discontinuation may lead to a disease flare.6.5Resistance to small molecule inhibitors can be divided into primary and secondary resistance.First, on‐target secondary mutations have been studied a lot, which spurs the enthusiasm for finding the next‐generation inhibitors with fewer resistance liabilities, such as ABL TKIs in CML, EGFR and ALK TKIs in lung cancer, and KIT TKIs in GISTs.In addition to traditional ways to address resistance, new methods which are capable of inducing loss of the total proteins, rather than just loss of their functions, are ideal for overcoming resistance caused by mutation or overexpression.6.6Combination therapy aims to increase and prolong the therapeutic benefits or reversal of primary and acquired resistance through the utility of agents with different mechanisms.First, vertical blockade of one signaling pathway is a strategy to increase the curative effect of small molecule inhibitors.p = 0.97).Second, small molecule inhibitors’ horizontal inhibition of two or more pathways is another schedule.p = 0.025) and was proved to be safe in phase III IMspire 150 trial.Combined small molecule inhibitors with ICIs is a prevailing direction.p = 0.807) and had a poor safety profile in previously untreated metastatic CRC.p = 0.016), warning that first‐generation EGFR TKIs cannot be continued when chemotherapy is initiated after disease progression.Combination with other traditional therapies is also prevalent, such as a CDK4/6 inhibitor plus an aromatase inhibitor for HR‐positive, HER2‐negative breast cancer, BCL‐2 inhibitor plus chemotherapy for AML, HDAC inhibitor plus radiotherapy for neuroblastoma.There are multiple ways to achieve combination therapy, but whether combination therapy is effective still needs to be verified by trials, especially large clinical trials, rather than a blind combination. Although clinical trials of combination therapy are at risk of failure, they are still a potential direction. We should continue researching combination therapies to improve patients’ prognoses.In summary, small molecule inhibitors bind to a wide range of targets to control the proliferation, survival, apoptosis, differentiation, metabolism, and TME of malignancies, which offer convenience and good curative effect to patients with tumors harboring specific gene alterations or patients with specific histological tumors.H. S. S. offered direction and guidance of the manuscript. G. H. L. and T. C. drafted the initial manuscript. X. L. M. revised the manuscript. G. H. L. and X. Z. illustrated the figures and tables for the manuscript. All authors approved the final manuscript.The authors have no conflict of interest to declare.No ethical approval was required for this study.https://clinicaltrials.gov/Supplementary Table S1 Summary of the pivotal candidates of small molecule kinase inhibitors. Data sources: Click here for additional data file.https://clinicaltrials.gov/Supplementary Table S2 Summary of the pivotal candidates of small molecule non‐kinase inhibitors. Data sources: Click here for additional data file.https://clinicaltrials.gov/Supplementary Table S3 Summary of the pivotal candidates of small molecule inhibitors targeting VEGFR.Data sources: Click here for additional data file.


Digital infrared thermal imaging (DITI), which detects infrared rays emitted from body surface to create a body heat map, has been utilized at various musculocutaneous conditions. Notably, DITI can demonstrate autonomic vasomotor activity in the nerve-innervated area, and thus may be of use in carpal tunnel syndrome (CTS). In this study, we compared DITI findings before and after carpal tunnel release (CTR) surgery in patients with unilateral CTS to investigate the corresponding neurophysiological changes.In this retrospective cohort study, DITI parameters such as the temperature differences between the median and ulnar nerve territories and median nerve-innervated digital anisometry were measured. Subjective symptom duration, pain scale, and ultrasonographic findings were also compared before and after CTR. Patients were evaluated before and 6 weeks after CTR, respectively.p = 0.003). The temperature differences between the median and ulnar nerve territories were not significantly changed. Subjective pain, the Simovic Weinberg Clinical Scale, and palmar bowing of the flexor retinaculum were also significantly improved .A total of 27 patients aged 59.0 ± 11.2 years were finally included. After CTR, median nerve-innervated thermal anisometry was improved (2.55 ± 0.96 °C to 1.64 ± 1.34 °C; Our results demonstrated that DITI findings could reflect an improvement in autonomic function after CTR. Therefore, DITI can be an objective method to assess pre- and post-operative neurophysiologic changes in CTS. Carpal tunnel syndrome (CTS) is one of the most common peripheral nerve entrapment syndromes which is caused by compression of the median nerve within the carpal tunnel . ElevateMost CTS patients with mild to modest symptoms receive conservative treatments. However, severe CTS patients or those with nerve lesions proved by electrodiagnosis often require surgery . Carpal Although there is no definitive standard for the diagnosis of CTS, electrodiagnostic testing is widely used in conjunction with clinical symptoms. In addition to physical examinations such as Tinel’s sign and Phalen’s maneuver, methods such as US, magnetic resonance imaging, and infrared body thermography are used –12. InfrSeveral studies using DITI have been published in previous studies on CTS; unfortunately, they did not show consistent results, –17 incluIn this context, we aimed to identify the thermographic, clinical, and sonographic changes before and after CTR in patients with unilateral CTS. Subsequently, we primarily investigated the significance of DITI in the evaluation and understanding of neurophysiological changes after CTR.This retrospective cohort study was performed from January 2020 to July 2021 and used a database from a single hospital’s electrodiagnostic laboratory. Symptoms, signs and laboratory findings were compared before and after surgery in patients who had undergone surgical treatment for unilateral CTS. All patients showed clinical and electrodiagnostic findings consistent with CTS, and significant symptoms that were difficult to improve with conservative treatment, such as pharmacological or injection treatment. Patients with diseases that may affect DITI; including central nervous system lesions involving the affected hand, lower cervical radiculopathy, systemic peripheral neuropathy, peripheral vascular disease, arthritis of the hand, previous history of hand or wrist surgery; and systemic diseases such as tumor, thyroid diseases, fibromyalgia, and uncontrolled diabetes; were excluded. The study was approved by the local institutional review board .The degree of pain was recorded through the numerical rating scale (NRS) before surgery, and Simovic Weinberg Clinical Scale  was usedUltrasonography was performed using a linear probe (12–5 MHz) in the proximal carpal tunnel between the pisiform and scaphoid bones. Patients were examined in a sitting position, with the elbow flexed at 90 degrees and the forearm fully supinated. The cross-sectional area and palmar bowing of the median nerve were measured at the site, respectively.The DITI test was performed according to the same protocol as previously published by these authors, and the same parameters were evaluated . The IRI= (digit 1 + digit 2 + digit 3)/3−digit 5Temperature difference between the median and ulnar nerve digits (ΔM−U digits)= thenar − hypothenar,Temperature difference between the thenar and hypothenar areas (Δthenar−hypothenar)= ΔM−U digits + Δthenar–hypothenar,Temperature difference between the median and ulnar nerve territories (ΔM−U territories)= │digit 1−digit 2│ + │digit 1−digit 3│ + │digit 2−digit 3│Median nerve-innervated digits anisometryWe designated six regions of interest of the thermal image of the palmar surface. Skin temperature was measured at the center of the finger pulp of the first, second, third, and fifth digits based on an area of 400 data points. Additionally, measurements were obtained from the thenar and hypothenar eminences based on an area of 800 data points. The mean value of all data points in the study area was determined. By translating the approved red-green-blue data to temperature, the final result was displayed in degrees Celsius. We created the indicators below for data interpretation and quantitative analysis. The values were calculated by substituting the average body temperature obtained within each region of interest.All patients underwent open CTR. The surgeon first made a 2 cm incision in the central part of the distal wrist. After dissecting the subcutaneous layer, the transverse carpal ligament was identified. Thereafter, decompression was performed by incision of the transverse carpal ligament while checking the median nerve sheath. Then, the surgeon removed adhesion tissues around the median nerve and confirmed that the nerve was sufficiently decompressed. Finally, each layer was sutured closed. Brachial plexus blockade was done prior to surgery.Except for electromyography, NRS, SCS, ultrasonography, and DITI were evaluated according to the same protocol by the same evaluator 6 weeks after surgery.Paired t-test was performed for each parameter before and after surgery. All statistical analyses were conducted using SPSS 22.0 .A total of 27 patients were included in this study; 7 men and 20 women with an average age of 59 ± 11.2 years. There were more participants with right hand than left hand involvement (right hand involvement: 59.3%). The average duration of symptoms was 12 ± 13.85 months. According to the electrodiagnostic severity grading, 3 patients were classified as moderate and 24 as severe; no patients were considered as mild (Table p <  0.001); and the Simovic Weinberg Clinical Scale changed from 25.44 ± 2.75 points before surgery to 6.67 ± 3.66 points (p <  0.001). Ultrasound findings of the median nerve cross-sectional area (CSA) in the proximal carpal tunnel was not significantly different from 0.20 ± 0.04 cm2 to 0.18 ± 0.08 cm2 before and after surgery, respectively (p = 0.191). However, a significant reduction in PB was observed, changing from 4.10 ± 0.71 mm to 3.24 ± 0.91 mm before and after surgery, respectively (p <  0.001)  , and that of the thenar and hypothenar areas before and after surgery. Other processed parameters, including ΔM − U digits, Δthenar−hypothenar, and ΔM − U territories, showed no statistically significant differences before and after surgery. Only median nerve-innervated digits anisometry showed a significant decrease after surgery compared to before surgery (2.55 ± 0.96 °C to 1.64 ± 1.34 °C; In this study, we demonstrated the decreased median nerve-innervated digital thermal ansiometry after CTR. This finding was in accordance with the postoperative improvement of clinical symptoms and US findings. In CTS, the increased interstitial fluid pressure in the carpal tunnel or the pressure that directly compresses the median nerve changes the microvasculature of the median nerve, resulting in ischemia and blood flow disorders . Damage Infrared thermographic changes were observed, along with pain reduction and clinical improvement after CTR in this study. It can be inferred that the statistically significant decrease in anisometry values after CTR in this study is due to the improvement of blood flow and autonomic function in the median nerve-innervated area after decompression of the carpal tunnel. On the other hand, there was no significant change in skin temperature other than anisometry after surgery. Specifically, secondary parameters, such as ΔM − U digits, Δthenar−hypothenar, and ΔM − U territories did not show significant changes after surgery. According to our previous study, these parameters, as opposed to anisometry, stand out in the early stage of the disease, but become less apparent as the severity and duration of the disease increases . Those wSince the median nerve contains most of the sympathetic nerves of the hand , it is tElectrodiagnostic testing is widely used in clinical practice as a tool to confirm CTS. However, while neuro-electrophysiological examination shows a strong correlation with the functional aspect of the median nerve, there are cases where there is no clear correlation with the symptoms of actual patients . MoreoveThis study has several limitations. This study is a retrospective study with single-center’s database and further validation studies are needed. Another objective test, an electrodiagnostic test, could not be performed after surgery, hence analysis thereof was not done. It unconfirmed whether there were other symptoms related to vasomotor function. Since DITI abnormalities are interpreted as being related to thin unmyelinated nerve fiber dysfunction, it is necessary to investigate whether there are other subjective complaints related to this in future studies.Carpal tunnel decompression surgery improved anisometry between median nerve-innervated digits in digital infrared thermal imaging, which is thought to be due to the improvement of vasomotor activity and blood flow mediated by the autonomic nervous system. This study suggests that digital infrared thermal imaging could possibility be of use as an objective evaluation method after carpal tunnel release.


BCKDHA, BCKDHB, and DBT that encode the E1α, E1β, and E2 subunits of the branched‐chain α‐ketoacid dehydrogenase (BCKD) complex. Various MSUD‐causing variants have been described; however, no structural rearrangements in BCKDHA have been reported to cause the classic MSUD phenotype. Here, we describe the classic patient with MSUD with compound heterozygous pathogenic variants in BCKDHA: a missense variant  and a paracentric inversion disrupting Intron 1 of BCKDHA, which was identified by whole‐genome sequencing and validated by fluorescence in situ hybridization. Using the sequence information of the breakpoint junction, we gained mechanistic insight into the development of this structural rearrangement. Furthermore, the establishment of junction‐specific polymerase chain reaction could facilitate identification of the variant in case carrier or future prenatal/preimplantation tests are necessary.Maple syrup urine disease (MSUD) is a rare autosomal recessive inherited disorder of branched‐chain amino acid metabolism caused by mutations in  BCKDHA.We report the classic form of maple syrup urine disease with paracentric inversion of Chromosome 19, which disrupts 1BCKDHA, BCKDHB, DBT, and DLD, respectively.BCKDHA consists of nine exons and is located on Chromosome 19q13.2.BCKDHA,BCKDHA, and present evidence derived from molecular tests. Furthermore, we showed that whole‐genome sequencing is a powerful tool for diagnosing genetic diseases because of its potential to detect pathogenic variants in coding and noncoding regions, copy number variations, and balanced structural rearrangements.Maple syrup urine disease  is a rare autosomal recessively inherited inborn metabolism error caused by deficiency in the branched‐chain alpha‐keto acid dehydrogenase (BCKD) complex.1.13− 10.9 mmol/l, BE −10.9 mmol/l) and hypoglycemia (glucose 1.8 mmol/l). She was immediately treated with glucose infusion and diet therapy, with restricted BCAA intake. The plasma levels of BCAA gradually decreased, and consequently, metabolic acidosis improved. Brain magnetic resonance imaging performed at 1 month of age showed no abnormalities. The plasma concentrations of BCAA in the patient were strictly monitored after discharge, and the leucine concentration was maintained at a safe level for MSUD treatment (150–400 nmol/ml). However, she had several metabolic decompensations triggered by infections and was admitted to the intensive care unit three times because of severe metabolic acidosis with impaired consciousness. Currently, she is 10 years of age. Her height and body weight were 141.6 cm  and 39.9 kg , respectively. The intellectual quotient (IQ) score was obtained using the Tanaka‐Binet Intelligence Scale . The patient's IQ score was 44. The patient was clinically and biochemically diagnosed with MSUD, and genetic testing was performed to confirm the diagnosis.The female patient was the third child of nonconsanguineous parents from Japan, with a birth weight of 2366 g. At birth, she was found to have mild hypoglycemia and was temporarily administered a glucose infusion till 5 days of age. She tested positive for MSUD in a newborn screening (NBS) performed at 5 days of age. The concentration of BCAAs in dried blood spot at NBS were markedly elevated with valine 776.7 nmol/ml , leucine 1646.7 nmol/ml , and isoleucine 526 nmol/ml . At 8 days of age, she was hospitalized and noted to have metabolic acidosis  to identify the cause of MSUD. We identified one heterozygous missense variant  in r Figure . Howevere Figure . ConsideA Figure . From thA Figure . ConfirmA Figure . Sanger A Figure . Based oA Figure . In addiA Figure . For theA Figure . AlthougA Figure , the proA Figure .2BCKDHA transcripts from the inversion allele, the inversion allele with disruption of Intron 1 possibly produces no protein at all owing to nonsense‐mediated mRNA decay. BCKD consists of three catalytic components, and it is considered that it cannot function well if one of the components changes significantly. Therefore, the present case exhibited a classic phenotype.Here, we describe a young female patient with MSUD caused by a compound heterozygous mutation, NM_000709.3:c.757G > A(p.Ala253Thr)/inv(19)(pter→q13.2::q13.32 → q13.2::q13.32 → qter). The c.757G > A (p.Ala253Thr) mutation was previously reported to result in a classic phenotype.The breakpoint junction of the inversion provides mechanistic insight into this genomic rearrangement. The blunt ends at Junction 1 and microinsertion of the unrelated sequence at Junction 2 implicate repair of double‐strand breaks (DSBs) via nonhomologous end joining. However, we identified GTGA microhomology at two nucleotides downstream of the putative breakpoint. Microhomology and microinsertion observed at the junction are the characteristics of the repair mechanism for DSBs or replication errors via alternative nonhomologous end joining; in other words, microhomology‐mediated end joining.In this case, we verified the presence of chromosome inversion by whole‐genome sequencing followed by junction‐specific PCR; however, FISH with probes encompassing the breakpoints was also a powerful tool to visually confirm the inversion. When the breakpoints of inversion are close to each other, confirming the inversion using ordinary metaphase FISH is difficult; thus, interphase FISH is often used for confirmation. However, with interphase FISH, information on chromosomal location is not available, and visually confirmation of the chromosomal structure of the chromosome is sometimes difficult. In this case, when one probe was designed at Breakpoint 1 and the other distal to Breakpoint 2, inversion was confirmed by signals on prometaphase chromosomes that had longer chromosome axes than at metaphase, providing high‐resolution FISH signals.BCKDHA. In recent years, low sequencing costs and rapid computing speed have created a standard genetic diagnosis environment for whole‐genome sequencing. Some reports recommend whole‐genome sequencing as the first approach in the diagnosis of unexplained diseases in newborns.BCKDHA.In this case, gene panel diagnosis revealed that the proband carried a pathogenic missense variant of maternal origin, and the RNA study led us to predict that another mutation of paternal origin was located in the noncoding region of Katsuyuki Yokoi: retrieved the data and drafted and revised the article. Yoko Nakajima: conception and design, analysis and interpretation, and drafting of the article. Yuta Sudo: provided chronic phase care for the patient. Tasuku Mariya: performed sequence analysis. Rie Kawamura: performed cytogenetic analysis. Makiko Tsutsumi: performed the eXome Hidden Markov Model. Hidehito Inagaki: performed whole‐exome. Tetsushi Yoshikawa: provided chronic phase care for the patient. Tetsuya Ito: provided neonetal period care for the patient. Hiroki Kurahashi: conception and design, analysis, interpretation, and critical revision of the article for important intellectual content.The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and the Helsinki Declaration of 1975, as revised in 2005 (5). The study protocol was approved by the Ethical Review Board for Human Genome Studies of Fujita Health University.Written informed consent for the publication of medical information and images was obtained from the patient's parents reported in this publication.


RL) is often assumed to constitute a fixed fraction of leaf dark respiration (RD) (i.e. a fixed light inhibition of respiration (RD)) and vary diurnally due to temperature fluctuations.Leaf daytime respiration . The light inhibition of RD varied diurnally and differed between climates and in trajectory between PFTs.The results highlight the entrainment of leaf daytime respiration to the diurnal cycle and that time of day should be accounted for in studies seeking to examine the environmental and biological drivers of leaf daytime respiration. The soil is a glacial outwash plain at both locations. In Australia, the measurements were conducted at the Hawkesbury Forest Experiment (HFE) site in Richmond, New South Wales  from 11 January 2020 to 29 February 2020 where the soil consists of a low‐fertility sandy loam , stomatal conductance for water vapour  and respiration in the dark were conducted on newly developed, fully expanded sun‐adapted leaves 0.1–2 m above ground level. Each leaf was measured 3–11 times throughout a single day, usually before sunrise until after sunset with a period of 35 min to 1 h in between each measurement. The measurements were conducted on at least four replicate plants (one leaf per plant) of each species under varying weather conditions . The leaves within species were selected based on morphological similarity.Measurements of leaf net CO2 leaf chamber and a 6800‐01A fluorometer set to 69% red and 31% blue light. On each measurement day, leaf temperature was preset to a constant temperature (To). For the measurements conducted in 2019 in Denmark To was set to match the median air temperature of the day based on weather forecasts, whereas To was set a few degrees below the maximum daily air temperature for the measurements in Australia in 2020 and Denmark in 2021. Hence, To varied between leaves both within and between all measured species but was kept constant within each leaf repeatedly measured throughout the day. The measurements were conducted using the Kok method with a relative air humidity of 35–80%, a flow rate of 300–350 μmol s−1, a fan speed of 10 000 rpm and a CO2 concentration of 410 ppm in the leaf chamber. The leaf irradiance response of Anet was measured from 100 μmol photons m−2 s−1 down to 0 μmol photons m−2 s−1 in steps of 10–12 irradiance levels. Before measuring Anet at each irradiance level, the reference and sample infrared gas analysers (IRGAs) were automatically matched, and gas exchange fluxes were given 2–5 min to stabilize. The stabilizing period for the 0 μmol photons m−2 s−1 irradiance level was increased to 10 min. Gas exchange fluxes were allowed to stabilize before initiation of the light response curve. This stabilization typically required 2–20 min.The measurements were conducted using a Li‐Cor 6800 portable photosynthesis system  with a 6 cmRL (μmol CO2 m−2 s−1) while RD (μmol CO2 m−2 s−1) was measured following leaf dark adaptation. The apparent RL was estimated based on the intercept of a linear regression fitted to the linear region above the Kok effect and RD was determined directly from the CO2 efflux in the dark. When using the Kok method, intercellular CO2 concentration  tends to increase as irradiance decreases, resulting in reduced photorespiration and increased carboxylation  was assumed to be 38.6 μbar for all species, and Γ* at any given leaf temperature (ΓleafT*) can be calculated according to Brooks & Farquhar 100. The rate of gross photosynthesis  was calculated as RL,To plus Anet at the 100 μmol photons m−2 s−1 irradiance level.The Kok method was used to calculate RL Fig. . Vj wasFarquhar :(Eqn 1)Farquhar :(Eqn 2)RL and RD exhibited diurnal variation measured at a constant temperature , all measurements of RL,To and RD,To were standardized by dividing each measurement from a single leaf by the mean RL,To or RD,To measurement of that leaf . Diurnal variations in RL,To/RL,To¯ and RD,To/RD,To¯, as well as the % light inhibition of RD,To were examined using generalized additive models (GAMs) with 95% pointwise confidence intervals fitted with automated smoothness selection in the mgcv library in R v.4.1.0  is a smooth function and εi is the residual error. This approach is nonparametric and makes no a priori assumption about the functional relationship between variables , within PFTs and within species. As the RL,To/RL,To¯, RD,To/RD,To¯ and the % light inhibition of RD,To measurements in Denmark were represented by nine deciduous tree species and one herbaceous species, the fitting of GAMs to PFTs was restricted to Australia. Significant diurnal variations of RL,To/RL,To¯, RD,To/RD,To¯ and the % light inhibition of RD,To were determined after computation of the first derivative (the slope) of the fitted GAMs with the finite differences method. The first derivatives were computed with 95% pointwise confidence intervals, and the trend was deemed significant when the derivative confidence interval was bounded away from zero at the 95% level  of n levels (k1 – kn). The variable kn,i is 1 if an observation is from level kn, and otherwise 0. The models explaining the highest variance with the minimum number of variables were identified using Akaike's information criterion (AIC).Model selection was used to determine whether the diurnal variation in Wieling, . Models Wieling,  and an iWieling,  were fitRL,To/RL,To¯, RD,To/RD,To¯ and the % light inhibition of RD,To from physiological and external environmental variables was examined using linear or logarithmic regression models with 95% pointwise confidence intervals. The ambient temperature (°C), light intensity (μmol m−2 s−1) and water vapour pressure deficit , provided by the climate station at HFE in Australia, were averaged for every hour during the time of measuring a light response curve of Anet. Model assumptions of normality and homoscedasticity of residuals were assessed and verified before analysis.The prediction of RL,To throughout a day, the GAMs that were initially fitted to the RL,To/RL,To¯ values were used to predict RL,To throughout a day based on a hypothetical scenario, where an RL,To measurement of 0.76 μmol CO2 m−2 s−1  was measured at 08:00 h. Accordingly, temporal GAMs at constant temperature were fitted to the RL,To/RL,To¯ values from Australia and Denmark using Eqn RL,To measurement of 0.76 μmol CO2 m−2 s−1 at other times of the day following:RL,Toi is the predicted rate of respiration at constant temperature at timei. RL,To is the measured RL,To (0.76 μmol CO2 m−2 s−1) at timex (08:00 h), εx is the residual error at timex and εi is the residual errors at timei. Null models (i.e. linear regression models without a slope) were fitted through the maximum, mean and minimum predicted values of the temporal GAMs for Australia and Denmark, and the predicted accumulated CO2 throughout a day was calculated for each model. The percentage predicted difference in accumulated CO2 between the maximum, mean and minimum models compared to the temporal GAMs was subsequently calculated. Eqn 10 model in order to calculate RL,T (i.e. RL under varying temperature conditions):RL,Ti changes for every 10°C temperature change, RL,Ti is the predicted rate of respiration at temperature Ti and To is the temperature at the time where RL,To was measured. Using Eqn RL,T (i.e. RL at different temperatures) was calculated at three different temperature profiles for both climates. A parametric approach with quadratic linear regression models and 95% pointwise confidence intervals was also constructed following:α is the model intercept, β and β1 are model coefficients, and ε indicates the residuals, which are assumed to follow a gaussian distribution. Using the guide in Dataset RL,To measurement from any time of the day can be used to predict RL,T or RL,To with either GAMs or quadratic linear regression models throughout the day depending on whether the daily temperature is constant or varying. The temperature profiles were derived from the ambient air temperature during the time of measuring the light response curves in Australia. Quadratic linear regression models were fitted through the maximum, mean and minimum temperature within 2 h intervals from 04:00 to 22:00 h from these data, yielding three different temperature profiles, respectively, which can be viewed in Fig. To examine the potential error of assuming a constant rate of RL,To and RD,To at the individual leaf level, RL,To/RL,To¯ and RD,To/RD,To¯ increased significantly from sunrise until morning or early midday, stabilized and then decreased significantly from late midday until sunset   Fig. , showingRL,To/RL,To¯ and RD,To/RD,To¯ persisted when analysing measurements within climates, and this pattern differed between the climates for both RL,To/RL,To¯ and RD,To/RD,To¯  and for the measurements collected in Denmark   while there was no significant association between Agross and the RD,To/RD,To¯ measurements from Denmark   and for the measurements in Denmark  while gsw was in general higher in Denmark  and in Denmark .There was a significant positive linear relationship between 05) Fig. . Agross05) Fig. . Agrossark Fig. . The ligark Fig. a  and RD,To/RD,To¯  measurements in Australia, such that when the ambient light intensity exceeded c. 100 μmol photons m−2 s−1 there was no apparent influence of the ambient light intensity on the RL,To/RL,To¯ and RD,To/RD,To¯ measurements . In addition, ambient temperature and VPD displayed no relationship with the RL,To/RL,To¯ and RD,To/RD,To¯ measurements  measured at 08:00 h was used to predict RL,To throughout the day at a constant temperature for both Australia and Denmark , as described in Eqn RL,To throughout the day were fitted to the maximum, mean and minimum RL,To values predicted by the temporal models  and the maximum, mean and minimum temperature profile models was 40, 57, 45 and 41 mmol m−2 d−1, respectively , or when pooling data across studies even when measurements are performed with the same temperature. Effects of time of day may result in variations of RL,To that in turn affect the calculation of photosynthetic parameters important for photosynthetic modelling. These could include estimates of maximum carboxylase (Vcmax) rates estimated with the one‐point Asat method, which has been shown to be sensitive to the chosen RL , may be erroneous. The light inhibition of RD,To showed only a weak association with the external light intensity above c. 100 μmol photons m−2 s−1 in Australia and decreased in a linear fashion with increasing Agross for both Australia and Denmark, as shown previously   by inserting the estimated model coefficients from Fig. RL follows a distinct relative diurnal pattern  regardless of environmental factors other than temperature as well as species and PFTs. Predictions should only be based on RL measurements derived with the Kok method.This study shows that time of day can have considerable effects on estimates of  Dataset  based onRL, RD and the light inhibition of RD exhibit significant diurnal variations when measured with a constant temperature in the field with the Kok method. The diurnal pattern of RL and RD differed in trajectory and magnitude between climates but not between PFTs, while it differed both between climates and in trajectory between PFTs for the light inhibition of RD. The results emphasize that time of day should be accounted for in studies seeking to estimate the response of leaf daytime respiration to variation in other factors (e.g. temperature and species), or when deriving inference across studies. The results highlight the dynamic nature of leaf daytime respiration that are driven by factors other than the measuring temperature and that RL and RD exhibit distinct diurnal patterns. Temporal variation in the regulatory relationship between physiological mechanisms and leaf respiration needs further attention to unveil the drivers of leaf respiration.This study demonstrates that AHF, DB, JY, KLG, MP and MGT planned and designed the research. AHF performed experiments, conducted field work and analysed the data. AHF and DB drafted the manuscript. All authors contributed to and revised the manuscript.Dataset S1 Excel spreadsheet containing the data used in this study including a guide on how to calculate RL,T throughout the day by taking into account the temporal variation in RL,To using the supplemented R script.Click here for additional data file.Fig. S1 Example of a light response curve of measured and Ci‐corrected leaf net CO2 exchange measurements plotted against photosynthetically active radiation.Fig. S2 Mean ambient temperature (°C) during the time of measuring the light response curves in Australia with fitted quadratic linear regression models depicting the maximum, mean and minimum temperature profiles.Fig. S3 Diurnal variation of leaf respiration in the light  and leaf dark respiration  from Australia and Denmark.Fig. S4 Leaf respiration in the light  and leaf dark respiration  measurements across Australia and Denmark plotted against the preset leaf measuring temperature during the time of measuring the light response curves of each leaf.Fig. S5 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯, of Solanum nigrum, Eucalyptus saligna, Eucalyptus tereticornis and Eucalyptus parramattensis from Australia with fitted generalized additive models.Fig. S6 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯, of Carya illinoinensis, Dichondra repens, Eucalyptus camaldulensis and Araujia sericifera from Australia with fitted generalized additive models.Fig. S7 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯, of Malus domestica, Liriodendron tulipifera and Platanus acerifolia from Australia with fitted generalized additive models.Fig. S8 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯,of Betula pendula, Quercus robur, Fraxinus excelsior and Salix cinerea from Denmark with fitted generalized additive models.Fig. S9 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯, of Alnus viridis, Alnus glutinosa, Helianthus annus and Corylus avellana from Denmark with fitted generalized additive models.Fig. S10 Diurnal variation of leaf respiration in the light, RL,To/RL,To¯, and leaf dark respiration, RD,To/RD,To¯, of Cornus sanguinea and Malus sylvestris from Denmark with fitted generalized additive models.Fig. S11Agross at 100 μmol photons m−2 s−1 irradiance conducted in Australia and Denmark plotted against gsw at 100 μmol photons m−2 s−1 irradiance.Fig. S12 Light inhibition of respiration measurements conducted in Australia and Denmark plotted against Agross at 100 μmol photons m−2 s−1 irradiance, and against the ambient light intensity.Fig. S13 Leaf respiration in the light  and leaf dark respiration  measurements conducted in Australia plotted against the recorded mean ambient temperature (°C) during the time of measuring the light response curves.Fig. S14 Leaf respiration in the light  and leaf dark respiration  measurements conducted in Australia plotted against the recorded mean ambient vapour pressure deficit (kPa) during the time of measuring the light response curves.Fig. S15 Temporal quadratic linear regression models of respiration in the light at constant temperature  and maximum, mean and minimum temperature variation models of respiration in the light at varying temperature  for Australia and Denmark, respectively.Notes S1 Supplementary site description: precipitation and temperature data from 2011 to 2021 and during the time of data collection for the region covering the Danish study sites and the study site in Australia.Click here for additional data file.Notes S2 R code to calculate temporal patterns of RL,T while accounting for temporal variations in RL,To.Table S1 Measured species in Denmark and Australia with individuals from three different plant functional types (PFTs) .Table S2 Total diurnal variation (%) of generalized additive models fitted to RL,To/RL,To¯, RD,To/RD,To¯ and % light inhibition of RD,To measurements in Fig. 1 that are driven by factors other than the measured temperature.Table S3 Model comparison between generalized additive models fitted across all RL,To/RL,To¯, RD,To/RD,To¯ or the light inhibition of RD,To measurements from Australia and Denmark (across climates) with time as the predictor variable (model 1) and a model where climate (across climates) or PFT (across PFTs) was added as a covariate (model 2) or an interaction term (model 3).Table S4 Mean and variation in the data points of the five light inhibitions of RD,To in Fig. 1.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries  should be directed to the Click here for additional data file.