Mutations In Tyrosine Kinase Domain Research Articles

Clinicopathologic and Molecular Characteristics of HER2 (ERBB2)-Altered Non–Small Cell Lung Cancer: Implications for Precision Medicine

The heterogeneous relationship between protein expression, amplification, and mutations in human epidermal growth factor receptor 2 (HER2) in non–small cell lung cancer (NSCLC) and the optimal methods for detecting these alterations remain unclear. We aimed to elucidate the clinicopathological and molecular characteristics of HER2-altered NSCLC and investigate practical approaches for identifying patients who might benefit from HER2-targeted therapies. Using next-generation sequencing data from 1680 individuals, we searched for patients with HER2-altered NSCLCs, including amplifications and mutations. Clinicopathological data and tissue slides were reviewed. Immunohistochemistry (IHC) and silver in situ hybridization were performed according to the American Society of Clinical Oncology/College of American Pathologists guidelines. Our analysis identified 89 (5.3%) patients with HER2-altered NSCLCs, comprising 30 (1.8%) with amplification and 59 (3.6%) mutations, and they were compared with 165 control patients. Of the 59 HER2-mutated cases, 52 harbored tyrosine kinase domain (TKD) mutations, primarily HER2 exon 20 insertions. HER2 TKD alterations were associated with younger age, female sex, nonsmoking status, adenocarcinoma with a micropapillary pattern, lung-to-lung metastasis, and poor overall survival. The 33 patients with TKD mutations and 3 with non-TKD point mutations showed incomplete or complete membranous HER2 immunoreactivity (1+ and 2+, 61.07%). Six patients exhibiting amplifications had an IHC score of ≤2+ despite their high copy numbers and concomitantly displayed other actionable EGFR, KRAS, SMARCA4, and other HER2 mutations. These HER2-altered NSCLCs with molecular coalterations showed heterogeneous patterns through HER2 IHC and silver in situ hybridization. Therefore, next-generation sequencing should be used to identify HER2 mutations in patients with NSCLC who present with concomitant alterations. In addition, the above clinicopathological characteristics and HER2 IHC results can be valuable determinants for identifying patients with HER2-altered NSCLC. These insights hold promise for the development of more effective diagnostic and therapeutic strategies for this complex subset of NSCLC patients.

Abstract CT284: A phase 3 randomized controlled trial of zongertinib (BI 1810631) compared with standard of care in patients with locally advanced or metastatic nonsquamous non-small cell lung cancer harboring HER2 tyrosine kinase domain mutations: Beamion LUNG-2

Abstract Background: First-line (1L) standard of care for patients with HER2 mutation-positive (HER2m+) non-small cell lung cancer (NSCLC) is platinum-based chemotherapy ± immunotherapy. To date, no targeted 1L treatments have been approved. Zongertinib is a HER2-selective tyrosine kinase inhibitor that binds to wild-type and mutated HER2, and spares EGFR. In Phase Ia of Beamion LUNG-1 (NCT04886804), zongertinib conferred objective response/disease control rates of 49/91% in patients with pretreated HER2 aberration-positive solid tumors, and 58/97% in those patients with HERm+ NSCLC, with manageable safety with few EGFR-associated adverse events. Purpose: Here we describe Beamion LUNG-2 (NCT06151574), a phase 3, randomized, controlled, open-label trial that will compare the efficacy and safety of 1L zongertinib with standard of care in patients with HER2m+, locally advanced/metastatic nonsquamous NSCLC. Experimental design: Approximately 270 patients will be randomized 1:1 to receive either zongertinib or standard of care. Key inclusion criteria: histologically/cytologically diagnosed advanced/metastatic nonsquamous NSCLC; no prior systemic treatment for locally advanced/metastatic disease; HER2 mutation in the tyrosine kinase domain; ≥1 measurable lesion (RECIST 1.1). Key exclusion criteria: tumors that have alterations with available therapy, and radiotherapy/major surgery ≤4 weeks prior to randomization. In the experimental arm, oral zongertinib 120 mg once daily will be given in 21-day cycles. In the comparator arm, intravenous pemetrexed 500 mg/m2 chemotherapy plus either cisplatin 75 mg/m2 or carboplatin area under the curve 5, plus pembrolizumab 200 mg will be given on day 1 every 3 weeks for 4 cycles, followed by pemetrexed 500 mg/m2 plus pembrolizumab 200 mg every 3 weeks for ≤35 cycles. In both arms, treatment will continue until progressive disease (RECIST 1.1), undue toxicity, or other criteria are met. Primary endpoint: progression-free survival (RECIST 1.1). Secondary endpoints: overall response (defined as best overall response of complete or partial response, RECIST 1.1), patient-reported outcomes (changes from baseline to Week 25), overall survival, and adverse events during the on-treatment period (CTCAE 5.0). Citation Format: Yi-Long Wu, Melissa Johnson, Ross Soo, Navid Baktash, Daniela Maier, Sabina Eigenbrod-Giese, Tatsuya Yoshida. A phase 3 randomized controlled trial of zongertinib (BI 1810631) compared with standard of care in patients with locally advanced or metastatic nonsquamous non-small cell lung cancer harboring HER2 tyrosine kinase domain mutations: Beamion LUNG-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT284.

Activating point mutations in the MET kinase domain represent a unique molecular subset of lung cancer and other malignancies targetable with MET inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas (PRCC). Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ~0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential, and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors.

EGFR exon 18 delE709_T710insD mutated stage IV non-small cell lung cancer treated with osimertinib: a case report.

Mutations in tyrosine kinase domain of epidermal growth factor receptor (EGFR) are observed in approximately 15% of non-small cell lung cancer adenocarcinoma. Exon 19 deletions or exon 21 L858R mutations are predominant in frequency and show high sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Exon 18 mutations are extremely rare and the delE709_T710insD mutation accounts for only 0.16% of mutations when occurring as a sole mutation. This specific mutation in exon 18 seems to respond to certain EGFR TKIs such as afatinib. However, given the rarity of this mutation, determining the most effective TKI for its treatment remains unclear. We report a 70-year-old woman diagnosed with stage IV-A lung adenocarcinoma harboring EGFR delE709_T710insD mutation treated in first-line with Osimertinib using standard schedule and doses experiencing renal toxicity and disease progression after 9 weeks of treatment.

A recurrent NTRK1 tyrosine kinase domain mutation pair is characteristic in a subset of dedifferentiated liposarcomas

IntroductionDedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes. Materials and Methods131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines. ResultsOut of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant. ConclusionsWe detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

102TiP A phase III randomised controlled trial of zongertinib (BI 1810631) compared with standard of care (SoC) in patients (pts) with locally advanced or metastatic non-squamous non-small cell lung cancer (NSCLC) harbouring HER2 tyrosine kinase domain (TKD) mutations: Beamion LUNG-2

102TiP A phase III randomised controlled trial of zongertinib (BI 1810631) compared with standard of care (SoC) in patients (pts) with locally advanced or metastatic non-squamous non-small cell lung cancer (NSCLC) harbouring HER2 tyrosine kinase domain (TKD) mutations: Beamion LUNG-2

BRCC36 associates with FLT3-ITD to regulate its protein stability and intracellular signaling in acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.

Gefitinib: Combination Therapy and Complex Delivery Systems (Review)

Introduction. The search for new methods of therapy for non-small cell lung cancer (NSCLC) is an urgent task of modern science. Gefitinib is a targeted drug widely used in the treatment of NSCLC in patients with a mutation in the epidermal growth factor receptor tyrosine kinase domain. However, using of gefitinib and other drugs from the group of tyrosine kinase inhibitors is to limited by rapidly developing resistance, for this reason finding of a ways overcome drug resistance is actual part of research interests.Text. The review is devoted to the use of gefitinib in modern developments: introduction to various targeted delivery systems (liposomes, micelles, microspheres, etc.), studying it in combination with other chemotherapeutic agents, as well as in combination with photo- or thermosensitive compounds in various micro- and nanostructured complexes.Conclusion. As a result of the analysis of literature data, it was shown that, despite the fact that gefitinib is a first-generation drug, foreign and Russian researchers consider it quite promising for further use in the treatment of NSCLC. At the same time, developments are being carried out both in the field of expanding combination therapy and in the field of creating complex structures of targeted action, into which, in addition to gefitinib, photosensitizers or other compounds with photo- or thermosensitive effects are introduced.

A Phase 1b Multi-Center Study of the FLT3 Inhibitor Gilteritinib in Combination with the IDH1 Inhibitor Ivosidenib or the IDH2 Inhibitor Enasidenib for Patients with Relapsed or Refractory Acute Myeloid Leukemia Who Have Co-Occurring FLT3/IDH1 or FLT3/IDH2 Mutations

Background: Mutations in isocitrate dehydrogenase isoenzymes 1 and 2 (IDH1 and IDH2) occur in 15- 25% of patients with acute myeloid leukemia. Enasidenib and ivosidenib, inhibitors of mutant IDH2 and IDH1 respectively, decrease intracellular levels of 2-HG, restore myeloid maturation, and lead to complete or partial remissions in approximately 40% of patients with IDH-mutant AML. While this response rate is remarkable in a relapsed and refractory AML population, nearly 60% of patients fail to respond. Approximately 30% of patients with IDH1/2 mutations have co-occurring mutations in the FMS like tyrosine kinase 3 (FLT3). Correlative science suggests that the acquisition of FLT3 mutations (either an internal tandem duplication (ITD) or tyrosine kinase domain (TKD)) is a common mechanism of resistance to IDH inhibitor monotherapy. Gilteritinib is a FLT3 inhibitor that targets the ITD and TKD mutations. This clinical trial (NCT05756777) will evaluate the safety and efficacy of concurrent IDH and FLT3 inhibition, while determining the maximum tolerated dose (MTD) for each of the combinations. Methods: NCT05756777 is a multi-center, open-label, experimental, interventional study evaluating the safety, tolerability, and preliminary efficacy of the combination of gilteritinib + ivosidenib (Cohort 1 - IDH1/FLT3 co-mutation) and gilteritinib + enasidenib (Cohort 2 - IDH2/FLT3 co-mutation) in patients with relapsed/refractory (R/R) acute myeloid leukemia with co-occurring FLT3 (ITD or TKD) and IDH1 or IDH2 mutations. Treatment with gilteritinib + ivosidenib will be administered for patients with IDH1/FLT3 co-mutations and gilteritinib + enasidenib will be administered for patients with IDH2/FLT3 co-mutations. Patients in both cohorts will dose daily throughout the 28-day cycle and without pause between cycles. The study will enroll 36 patients with relapsed/refractory AML with co-occurring FLT3 (ITD or TKD) and IDH1 or IDH2 mutations. The dose escalation schema will use the Bayesian Optimal Interval (BOIN) design. This study is open for accrual at Memorial Sloan Kettering Cancer Center. The study population will include patients with AML with dual FLT3 and IDH1/IDH2 mutations R/R to initial intensive induction therapy, or for patients treated with low intensity therapy, the patient must be refractory to treatment with a single agent hypomethylating agent (HMA) or low dose cytarabine (LDAC) (at least two cycles) or an HMA/LDAC in combination with venetoclax (at least one cycle) or another standard of care therapy (e.g. gemtuzumab ozogamicin, glasdegib/LDAC). Patients with a history of autologous or allogeneic stem cell transplant for AML are permitted to participate in the study. This study is partially supported by Astellas Pharma Global Development, Inc. Clinical trial information: https://classic.clinicaltrials.gov/ct2/show/NCT05756777

Novel Insights in TKI Resistance in BCR:: ABL1-Positive B-Cell Acute Lymphoblastic Leukemia Beyond Kinase Domain Mutations

Background and Aim Despite the introduction of tyrosine kinase inhibitors (TKIs), approximately 30% of newly diagnosed pediatric and adult BCR:: ABL1-positive B-cell acute lymphoblastic leukemia (B-ALL) patients experience relapsed or refractory disease (Biondi et al, Lancet Haematology 2018; Bassan et al, Journal of Clinical Oncology 2010). ABL1 kinase domain mutations, hindering TKI binding, explain part of the relapses and are acquired during TKI therapy but are rarely detected in newly diagnosed BCR:: ABL1-positive B-ALL patients (Baer et al, Haematologica 2022). Intrinsic TKI resistance mechanisms in BCR:: ABL1-positive B-ALL present prior to TKI treatment are less well documented. Therefore, we aimed to identify potential biomarkers for TKI sensitivity at diagnosis using primary patient samples and newly generated cell lines made from patient-derived xenografts (PDX cell lines). Patients and Methods Leukemic cells from diagnosis of BCR:: ABL1-positive B-ALL patients without ABL1-kinase domain mutations were used to measure cell survival in an ex vivo co-culture assay after imatinib (1.95-125 μM), dasatinib (1.95-125 nM), or bosutinib (0.016-2 μM) exposure. TKI sensitivity was assessed using the area under the dose-response curve (AUC), with <80 arbitrary units (AU) classified as imatinib-sensitive, 80-120 AU as intermediate, and >120 AU as resistant. Multiplex ligation-dependent probe amplification, whole exome sequencing, and RNA sequencing were performed on diagnostic material if available. ABL1 phosphorylation and expression levels were measured using (phospho)flow cytometry in patient-derived xenografts from diagnostic samples and (PDX) cell lines. Results We determined ex vivo sensitivity to imatinib, dasatinib, and bosutinib in 32 pediatric and 19 adult BCR:: ABL1-positive B-ALL patient samples. We observed a substantial variability in ex vivo imatinib response in pediatric and adult patient samples (median AUC: 98 AU; range: 16 to 281 AU). The sensitivity towards imatinib strongly correlated with the sensitivity towards second-generation TKIs dasatinib and bosutinib (rho = 0.84; p < 0.001 and rho= 0.89; p <0.001). Moreover, imatinib's AUC of samples derived from newly diagnosed patients who relapsed after TKI treatment was higher compared with those derived from patients who did not suffer from a relapse (median AUC 123 vs 84 AU; p = 0.03). We observed that an increase in ex vivo imatinib resistance was correlated with a decreased amount of phosphorylated ABL1 protein (rho=-0.92; p=0.0013) and total ABL1 protein (rho=-0.82; p=0.01) in untreated, newly diagnosed samples. Ex vivo exposure to imatinib reduced the ABL1 phosphorylation levels in both sensitive and resistant samples but resistant cells exhibited lower levels of (p)ABL1 protein, indicating reduced dependence on BCR-ABL1 signaling. The ex vivo imatinib resistance in samples carrying deletions in B-cell development genes IKZF1 and/or PAX5 was higher than in samples without these deletions (median AUC: 122 versus 80 AU; p=0.01). In addition, one imatinib-resistant patient had a p.His1038Arg mutation in ZEB2, which is recurrent in B-ALL and this gene has also been described as B-cell development gene (Studd et al, Blood Cancer Journal 2021). Interestingly, another resistant patient had a mutation in SETD2,a gene previously associated with ex vivo TKI resistance in chronic myeloid leukemia cell lines (Sheng et al, Cell Proliferation 2019). Other secondary lesions included SH2B3 inactivation lesions and CRLF2 rearrangements. These lesions were present in intermediately sensitive and resistant BCR:: ABL1-positive samples and are expected to lead to increased JAK/STAT signaling, potentially serving as a resistance mechanism for TKI. Conclusion Our study revealed that the amount of total and phosphorylated ABL1 protein was correlated with ex vivo TKI sensitivity. Newly diagnosed TKI-resistant BCR:: ABL1 samples were further characterized by somatic lesions in B-cell development genes including IKZF1. These results link the known poor prognostic value of IKZF1 deletions in BCR:: ABL1-positive B-ALL patients (van der Veer et al. Blood 2014; Fedullo et al, Haematologica 2019) with intrinsic mechanisms of TKI resistance other than tyrosine kinase domain mutations.

Mutation of Epigenetic Regulators at Diagnosis Is an Independent Predictor of Tyrosine Kinase Inhibitor Treatment Failure: A Report from the Residiag Study

Introduction Additional mutations at chronic myeloid leukemia (CML) diagnosis have been shown to variably affect tyrosine kinase inhibitor (TKI) response and were inconstantly detected at loss of response. Contradictory observations may have resulted from difficulties in reliably inferring CML clonal architecture from mutations quantified by NGS, BCR::ABL1 by qRT-PCR, ABL1-TK by RNA-Seq. The RESIDIAG study was designed to identify at diagnosis molecular markers predictive of TKI treatment failure and analyze the clonal dynamics using asymmetric capture sequencing (aCAP-Seq). Methods DNA samples from 60 TKI responders (median follow-up: 7.1 years) were sequenced at diagnosis by aCAP-Seq, allowing quantification and breakpoint sequencing of genomic BCR::ABL1 fusion together with 43 myeloid genes, including ABL1 with a limit of detection of ~0.1% for BCR::ABL1 and 0.5-1% for SNVs and Indels. In parallel, CML patients who experienced treatment failure (ELN 2020) were sequentially analyzed at diagnosis and at failure (n=53), at diagnosis only (n=1), and at failure only (n=3). Small deletions and BCR::ABL1 breakpoints were analyzed to search for microhomology-mediated end-joining (MMEJ) repair signatures. Mutation analysis followed strict medical standards. Results The proportion of patients receiving imatinib at diagnosis wasn't statistically different (p=0.08; Pearson's Chi-square test) between responders (70%) and the failure group (56%). At diagnosis, the number of mutations was higher (p<0.001, t-test with Welch correction) in the failure group (0.9±0.1) as compared with responders (0.18± 0.05). ASXL1, DNMT3A, and TET2 were more frequently mutated at diagnosis in the failure group (38.6%, p<0.001; 10.5%, p=0.01, and 4.3%, p=0.025 respectively; Pearson's Chi²test) as compared with responders (6.7%, 0% and 0% respectively) and the presence of a mutation in one of these genes was highly associated with a reduced failure-free survival (p<0.001, Log-ranked test, Figure 1). An MMEJ signature at BCR::ABL1 breakpoints was more frequent (p=0.014, Pearson's Chi² test) in the failure group (29.1%) than in the responder group (9.1%). and was significantly associated with the ELTS score (p=0.005, Pearson's Chi² test) but not the Sokal score (p=0.017, Pearson's Chi² test). At first failure, 69% of patients had additional mutations in ASXL1 (39%), ABL1-TK (38%), DNMT3A (18%), or TET2 (15%), while other genes were rarely (<5%) mutated. ASXL1 mutations found at diagnosis were still detectable in 18/22 patients at failure and VAF suggested that double mutant BCR::ABL1/ ASXL1 clones were driving TKI failure with or without BCR:: ABL1 tyrosine kinase domain mutation (TKD). ASXL1 mutations disappearing upon treatment were mostly at low frequency <5% at diagnosis and thus possibly not clonally related to CML. Furthermore, a stepwise selection of variables followed by multivariate analysis with the Fine-Gray model identified the age of patients (sdHR [95% C.I.] = 0.99 [0.97-1.00]; p=0.08) and the presence of a mutation in epigenetics regulators at diagnosis as factors affecting the onset of TKD mutations (sdHR = 3.01 [1.76-5.15]; p<0.001). Intriguingly, co-occurring mutations in DNMT3A and TET2 were found in five patients at TKI failure. Their allelic frequency kinetics suggested that these belonged to the same clone driving CML relapse in 4/5 CML patients, while one patient also had a TKD mutation likely involved in CML relapse. Finally, multivariate COX regression analysis identified two independent predictors of TKI failure at diagnosis: a high-risk ELTS score (p=0.001; hazard ratio=3.42 [1.62-7.22] with low-risk as baseline) and a mutation in ASXL1, DNMT3A or TET2 (p=0.002; hazard ratio=2.87 [1.49-5.51]). Conclusion Altogether these results strongly point to the contribution of epigenetic regulator mutations in the emergence of TKI failure in CML and warrant further biological studies to better understand the underlying mechanisms

OA19.05 Activating MET Tyrosine Kinase Domain Mutations as de Novo Oncogenic Drivers in Non-small Cell Lung Cancer

OA19.05 Activating MET Tyrosine Kinase Domain Mutations as de Novo Oncogenic Drivers in Non-small Cell Lung Cancer

A T cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo

Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34− AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.

Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors.

ROS1 is the largest receptor tyrosine kinase in the human genome. Rearrangements of the ROS1 gene result in oncogenic ROS1 kinase fusion proteins that are currently the only validated biomarkers for targeted therapy with ROS1 TKIs in patients. While numerous somatic missense mutations in ROS1 exist in the cancer genome, their impact on catalytic activity and pathogenic potential is unknown. We interrogated the AACR Genie database and identified 34 missense mutations in the ROS1 tyrosine kinase domain for further analysis. Our experiments revealed that these mutations have varying effects on ROS1 kinase function, ranging from complete loss to significantly increased catalytic activity. Notably, Asn and Gly substitutions at Asp2113 in the ROS1 kinase domain were found to be TKI-sensitive oncogenic variants in cell-based model systems. In vivo experiments showed that ROS1 D2113N induced tumor formation that was sensitive to crizotinib and lorlatinib, FDA-approved ROS1-TKIs. Collectively, these findings highlight the tumorigenic potential of specific point mutations within the ROS1 kinase domain and their potential as therapeutic targets with FDA-approved ROS1-TKIs.

CML with De Novo NPM1 Mutation and Rapid Progression to Myeloid Blast Cri-Sis despite Treatment with Imatinib: A Case Report

In this case we present a 41 year old male with chronic myeloid leukaemia who despite scoring low risk for Sokal progressed rapidly to blast crisis without warning and after having achieved a partial response to Imatinib therapy. We investigated using retrospective Next Generation Sequencing (NGS) and identified a novel NPM1 mutation at diagnosis and blast crisis together. We also found tyrosine kinase domain mutations using NGS not found with Sanger sequencing. We believe this case highlights the effectivity of NGS in identifying novel and existing mutations with higher sensitivity, which could help improve existing practice in prognostication and management of CML disease in patients not responding to treatment adequately.

Rational design of 4-((6-phenoxypyrimidin-4-yl)amino)-N-(4-(piperazin-1-yl)phenyl)-1H-pyrazole-3-carboxamide (LT-540-717) as orally bioavailable FLT3 inhibitor

In recent years, fms-like tyrosine kinase 3 (FLT3) was confirmed as an exciting target for treatment of AML. However, resistance to FLT3 inhibitors caused by acquired point mutations in tyrosine kinase domain (TKD) have limited their sustained efficacious. Thus, there remains an unmet need to develop high-efficacy FLT3 inhibitors against both FLT3 internal tandem duplication (ITD) and FLT3 (TKD) mutations. Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC50: 0.62 nM), starting from FN-1501. Compound 32 exhibited highly inhibitory activity against several acquired FLT3 mutations including FLT3 (ITD, D835V), FLT3 (ITD, F691L), FLT3 (D835Y) and FLT3 (D835V). Additionally, 32 displayed potent antiproliferative activity against FLT3-mutation driven BaF3 and AML cells. Oral administration of 32 (25 mg/kg, QD) significantly prohibited tumor growth (tumor-inhibition rate is 94.18%), and no obvious side effect was observed even when increasing dose to 50 mg/kg (tumor-inhibition rate is 93.98%). Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T1/2 = 3.5 h in rat and T1/2 = 11.1 h in beagles), and a satisfactory metabolic stability. In summary, these results show the therapeutic potential of 32 to become a new anti-AML drug, especially for AML harboring dual FLT3 (ITD, TKD) mutations.

Abstract 4009: Discovery of PLM-102, a highly potent 3rd generation FLT3 inhibitor, in drug-resistant FLT3-ITD-TKD mutated acute myeloid leukemia

Abstract Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases and FLT3-ITD mutations are associated with a particularly unfavorable prognosis, with an increased risk of relapse and shorter overall survival (OS) compared with AML patients without the mutation. Even though multiple FLT3 inhibitors are in various stages of clinical evaluation, resistance to FLT3 inhibitors resulting from acquired point mutations in tyrosine kinase domain (TKD) have limited the sustained efficacy of treatments. Among them, a “gatekeeper” mutation (F691L) is resistant to most available FLT3 inhibitors. Here, we show the efficacy and mechanism of PLM-102, developed an orally available and highly selective small molecule, in overcoming drug-resistant FLT3-ITD-TKD mutations as a strong candidate of the 3rd generation of FLT3 inhibitor. PLM-102 displays extraordinary potency of enzyme inhibitory activities of all FLT3 WT/mutants including F691L with IC50 values of <1 nM. In FLT3-ITD-positive cell lines (MV4-11, MOLM-13, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), PLM-102 shows about 18~25-fold higher anti-proliferative activities than Gilteritinib. Also, PLM-102 potently inhibited the phosphorylation of FLT3 and its downstream signaling pathways, and induced apoptosis as evidenced by PARP-cleavage and caspase-3 activation. Moreover, in AML-related xenograft mouse models (MV4-11, MOLM-14, Ba/F3-ITD-D835Y, Ba/F3-ITD-F691L), once-daily oral dose of PLM-102 significantly suppressed the tumor growth and achieved complete tumor regression. In addition, the outstanding efficacy of our compound has been confirmed in large tumor xenograft models. These results are very encouraging because large tumors have a limited blood supply and high interstitial fluid pressure, leading to a poor absorption of anticancer drugs. Pharmacokinetics of PLM-102 displayed higher bone-marrow exposure indicating better target engagement and benefits in the clinical translation in AML. In conclusion, PLM-102 is a promising therapeutic candidate for the FLT3-ITD-mutated AML as well as the acquired resistance to current FLT3 inhibitors. Citation Format: Jin-Hee Park, Jae-Seon Lee, Su-jin Oh, So-Deok Lee, Yong June Choi, Keon Wook Kang, Miran Moon, Soo Yeon Jang, Myung Jin Kim, Yong-Chul Kim. Discovery of PLM-102, a highly potent 3rd generation FLT3 inhibitor, in drug-resistant FLT3-ITD-TKD mutated acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4009.

Abstract 1062: A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme

Abstract AML is the most common acute leukemia in adults over 50 years of age. Current therapeutic approaches are focused on high or low dose chemotherapy in the front line setting, with use of molecularly targeted agents in unfit patients or the relapsed/refractory setting. However, routine genomic profiling for common molecular lesions (e.g. FLT3 mutation) incompletely stratifies the target patient population, and more personalized and comprehensive approaches could be of significant clinical benefit. To this end, we have developed an in vitro functional genomics platform for the systematic characterization of gene dependency on drug targets using CRISPR/Cas9 technology. Methods: Retrospective pre-therapy samples were obtained for a cohort of AML patients who subsequently went onto therapy with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g. GCLAM). Unsorted primary tumor cells from these samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library. This customized sgRNA library (Fx Heme) was designed to inhibit expression of genes encoding the targets of all FDA-approved oncology drugs, as well as internal positive and negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Dependency on targets of sorafenib was compared to clinical outcome (complete response (CR) without minimal residual disease (MRD), incomplete response, CR with MRD, or refractory) following treatment with sorafenib plus chemotherapy. Results: Among the 22 AML patients analyzed, 14 reached CR and 8 were classified as incomplete responders (refractory or MRD). Gene dependency on sorafenib targets including FLT3, KIT, PDGFR, and all RAF isoforms was frequently observed (12/22 cases). Among cases indicating any sorafenib target gene dependency, 5/12 (42%) demonstrated dependence on 2 or more sorafenib target genes. Overall, Fx Heme achieved 78.6% sensitivity, 87.5% specificity, and 91.7% PPV for prediction of observed clinical outcome. In contrast, stratification based on FLT3 mutation (internal tandem duplication or tyrosine kinase domain mutation) demonstrated 64.3% sensitivity, 50% specificity, 69.2% PPV. In summary, our results establish feasibility of CRISPR-based Fx Heme comprehensive functional genomic profiling for AML precision medicine, and validate this approach in a retrospective study cohort. Functional genomics may contribute to more effective personalization of AML treatment by uncovering drug target dependencies not readily identified by conventional genomic profiling, in an unbiased, comprehensive and patient-specific fashion. Citation Format: Stephane Corbel, Karl Hodel, Kathleen Tran, Maci Meyers, Jing Tong, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath. A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1062.

A customized mass array panel for BCR::ABL1 tyrosine kinase domain mutation screening in chronic myeloid leukemia

IntroductionThe therapeutic strategy and management of chronic myeloid leukemia (CML) have rapidly improved with the discovery of effective tyrosine kinase inhibitors (TKIs) to target BCR::ABL1 oncoprotein. However, nearly 30% of patients develop TKI resistance due to acquired mutations on the tyrosine kinase domain (TKD) of BCR::ABL1. MethodsWe customized a mass array panel initially intended to detect and monitor the mutational burden of hotspot BCR::ABL1 TKD mutations accumulated in our database, including key mutations recently recommended by European LeukemiaNet. Additionally, we extended the feasibility of using the assay panel for the molecular classification of myeloproliferative neoplasms (MPNs) by incorporating primer sets specific for analyzing JAK2 V617F, MPL 515 K/L, and CALR types 1 and 2. ResultsWe found that the developed mass array panel was superior for detecting and monitoring clinically significant BCR::ABL1 TKD mutations, especially in cases with low mutational burden and harboring compound/polyclonal mutations, compared with direct sequencing. Moreover, our customized mass array panel detected common genetic alterations in MPNs, and the findings were consistent with those of other comparable assays available in our laboratory. ConclusionsOur customized mass array panel was practicably used as a routine robust assay for screening and monitoring BCR::ABL1 TKD mutations in patients with CML undergoing TKI treatment and feasible for analyzing common genetic mutations in MPNs.

Mutations in the MET tyrosine kinase domain and resistance to tyrosine kinase inhibitors in non-small-cell lung cancer

BackgroundThe Mesenchymal epithelial transition factor (MET) gene encodes a receptor tyrosine kinase with pleiotropic functions in cancer. MET exon 14 skipping alterations and high-level MET amplification are oncogenic and targetable genetic changes in patients with non-small-cell lung cancer (NSCLC). Resistance to tyrosine kinase inhibitors (TKIs) has been a major challenge for targeted therapies that impairs their clinical efficacies.MethodsEighty-six NSCLC patients were categorized into three cohorts based on the time of detecting MET tyrosine kinase domain (TKD) mutations (cohort 1: at baseline; cohort 2: after MET-TKI treatment; cohort 3: after EGFR-TKI treatment). Baseline and paired TKI treatment samples were analyzed by targeted next-generation sequencing.ResultsMET TKD mutations were highly prevalent in METex14-positive NSCLC patients after MET-TKI treatment, including L1195V, D1228N/H/Y/E, Y1230C/H/N/S, and a double-mutant within codons D1228 and M1229. Missense mutations in MET TKD were also identified at baseline and in post-EGFR-TKI treatment samples, which showed different distribution patterns than those in post-MET-TKI treatment samples. Remarkably, H1094Y and L1195F, absent from MET-TKI-treated patients, were the predominant type of MET TKD mutations in patients after EGFR-TKI treatment. D1228H, which was not found in treatment-naïve patients, also accounted for 14.3% of all MET TKD mutations in EGFR-TKI-treated samples. Two patients with baseline EGFR-sensitizing mutations who acquired MET-V1092I or MET-H1094Y after first-line EGFR-TKI treatment experienced an overall improvement in their clinical symptoms, followed by targeted therapy with MET-TKIs.ConclusionsMET TKD mutations were identified in both baseline and patients treated with TKIs. MET-H1094Y might play an oncogenic role in NSCLC and may confer acquired resistance to EGFR-TKIs. Preliminary data indicates that EGFR-mutated NSCLC patients who acquired MET-V1092I or MET-H1094Y may benefit from combinatorial therapy with EGFR-TKI and MET-TKI, providing insights into personalized medical treatment.