Mutations In Tyrosine Kinase Domain Research Articles

Safety, Efficacy and Biomarker Analysis of Crizotinib in MET Mutated Non-Small Cell Lung Cancer - Results from the Drug Rediscovery Protocol.

MET mutations occur in 3-4% of advanced non-small cell lung cancer (aNSCLC), correlating with poor survival. Despite known sensitivity of MET mutated (METmut) aNSCLC to c-MET-inhibition, no approved therapies existed until 2022. In the Drug Rediscovery Protocol (NCT0295234), patients with an actionable molecular profile are treated with off-label registered drugs. Both treated and untreated patients with aNSCLC harboring MET exon 14 skipping (METex14) or other METmuts received crizotinib 250 mg BID until disease progression or intolerable toxicity. Primary endpoints were clinical benefit (CB: RECIST v1.1 confirmed partial response (PR), complete response (CR) or stable disease (SD) ≥16 weeks) and safety. Patients were enrolled using a Simon-like two-stage design, with eight patients in stage 1 and if ≥1/8 patients had CB, 24 patients in stage 2. Whole genome and RNA-sequencing were performed on baseline biopsies. Between 09/2018 and 10/2022, 30 patients started treatment, and 24 were response-evaluable after completing ≥1 full treatment cycle. Two patients (8.3%) achieved CR, thirteen (54.2%) PR and two (8.3%) SD. The CB-rate was 70.8% (95%CI 48.9-87.4) and the objective response rate was 62.5% (95%CI 40.6-81.2). After 21.2 months median follow-up, median duration of response, progression-free and overall survival were 9.3 (95%CI 6.5-NA), 10.2 (95%CI 6.0-20.1) and 13.0 months (95%CI 9.0-NA), respectively. Twenty-three treatment-related grade ≥3 adverse events occurred in 12/30 patients (40%), causing treatment-discontinuation in three (10%). One patient (achieving CR) had a tyrosine kinase domain mutation (p.H1094Y), all other patients had METex14. Crizotinib is a valuable treatment option in METmut aNSCLC.

Importance of PTM of FLT3 in acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic cells. Internal-tandem duplication domain (ITD) mutation and tyrosine kinase domain (TKD) mutation are the two most common mutations in acute myeloid leukemia (AML). Post-translational modifications (PTMs) of FLT3, such as glycosylation and ubiquitination, have been shown to impact various aspects of the protein in both wild-type (WT) and mutant forms of FLT3. In this review, we describe how the glycosylation status of FLT3 affects its subcellular localization, which significantly impacts the activation of downstream signaling, and the impact of specific ubiquitination on FLT3 function and stability, which may be associated with disease progression. Moreover, potential novel therapeutic strategies involving a combination of FLT3 tyrosine kinase inhibitors and drugs targeting glycosylation or ubiquitination are discussed.

The multi-kinase inhibitor CG-806 exerts anti-cancer activity against acute myeloid leukemia by co-targeting FLT3, BTK, and aurora kinases

Despite the development of several Fms-like tyrosine kinase 3 (FLT3) inhibitors that have improved outcomes in patients with FLT3-mutant acute myeloid leukemia (AML), drug resistance is frequently observed, which may be associated with the activation of additional pro-survival pathways, such as those regulated by BTK, aurora kinases (AuroK), and potentially others, in addition to acquired tyrosine kinase domain (TKD) mutations of FLT3 gene. FLT3 may not always be a driver mutation. We evaluated the anti-leukemia efficacy of the novel multi-kinase inhibitor CG-806, which targets FLT3 and other kinases, to circumvent drug resistance and target FLT3 wild-type (WT) cells. The anti-leukemia activity of CG-806 was investigated by measuring apoptosis induction and analyzing the cell cycle using flow cytometry in vitro. CG-806 demonstrated superior anti-leukemia efficacy compared to commercially available FLT3 inhibitors, both in vitro and in vivo, regardless of FLT3 mutational status. The mechanism of action of CG-806 may involve its broad inhibitory profile against FLT3, BTK, and AuroK. In FLT3 mutant cells, CG-806 induced G1 phase blockage, whereas in FLT3 WT cells, it resulted in G2/M phase arrest. Targeting FLT3 and Bcl-2 and/or Mcl-1 simultaneously results in a synergistic pro-apoptotic effect in FLT3 mutant leukemia cells. The results of this study suggest that CG-806 is a promising multi-kinase inhibitor with anti-leukemic efficacy regardless of FLT3 mutational status. A phase 1 clinical trial of CG-806 for the treatment of AML has been initiated (NCT04477291). Key points The multi-kinase inhibitor CG-806 exerts superior anti-leukemic activity in AML, regardless of its FLT3 status. CG-806 triggered G1 arrest in FLT3 mutated cells and G2/M arrest in FLT3 WT cells through the suppression of FLT3/BTK and aurora kinases. Concomitantly targeting FLT3 and Bcl-2 and/or Mcl-1 exerted synergistic pro-apoptotic effects on both FLT3 WT and mutated AML cells.

Selpercatinib in non-MTC, RET-mutated tumors: Efficacy in MEN-associated and other tumors.

3150 Background: Selpercatinib, a first-in-class highly selective and potent RET kinase inhibitor with CNS activity, is approved in multiple countries for the treatment of RET-activated cancers. Current indications for RET mutant disease are limited to MTC. This analysis examined if selpercatinib was effective in non-MTC, RET mutation positive tumors and whether certain RET mutations demonstrated better activity (n=23; data cut-off: 13Jan2023). Methods: The phase 1/2 LIBRETTO-001 trial (NCT03157128) enrolled pts with locally advanced/metastatic RET-altered solid tumors, including pts with RET mutations in tumors other than MTC. Mutations must be known to be activating based on activity in MTC or other published evidence. Primary endpoint was objective response rate (ORR) by independent review committee (IRC). Secondary endpoints included ORR by investigator (INV), duration of response (DoR), progression-free survival (PFS), and safety. Results: Fourteen different tumor types were identified among the 23 pts with non-MTC RET-mutated tumors. This included 8 tumors from the Multiple Endocrine Neoplasia (MEN) syndrome spectrum; adrenal (pheochromocytoma; n=5), paraganglioma (n=2), neuroendocrine (n=1) (Table). Most of the mutations were located in extracellular cysteine-rich domain (CRD) and in the tyrosine kinase domain (TKD) of RET. In 23 efficacy-evaluable pts, confirmed ORR by IRC was 21.7% (5/23, 95% CI: 7.5, 43.7). ORR by IRC for pts with MEN-associated tumors (n=8) was 37.5% while five of these pts (62.5%) had stable disease lasting at least 16 weeks (SD16+). ORR by IRC for the sub-population with pheochromocytoma (n=5) was 40.0% and for pts with tumors manifesting RET extracellular cysteine mutations (n=5) was 60.0%. Of the 15 pts with non-MEN-associated tumors, 2 pts (8.7%; mutation in TKD) had a partial response (both with NSCLC adenocarcinoma, of which one had co-existent MTC). For the overall pts, the median DoR was 12.2 months (95% CI: 3.8, NE) and median PFS was 5.5 months (95% CI: 1.8, 19.4). No new safety signals were identified compared to previous reports. One grade 5 AE, general physical health deterioration, was observed and deemed by INV as not related to selpercatinib. Conclusions: This study suggests that RET-mutated, MEN-associated cancers (adrenal, paraganglioma, neuroendocrine) may benefit from selpercatinib treatment, which could be mediated through mutations in the extracellular CRD. The non-MTC RET-mutated tumor types appear not to derive benefit from selpercatinib. Clinical trial information: NCT03157128 . [Table: see text]

IAM1363-01: A phase 1/1b study of a selective and brain-penetrant HER2 inhibitor for HER2-driven solid tumors.

TPS3186 Background: HER2 activation promotes carcinogenesis, prompting the development of HER2-directed therapies in cancers with HER2 alterations. There is a critical need for novel agents to treat HER2-driven cancers, as current inhibitors display significant toxicity and lack efficacy against certain mutations. IAM1363 (formerly IAM-H1 and ENT-H1) is an irreversible and brain-penetrant small molecule inhibitor targeting both wildtype HER2 and oncogenic HER2 mutants, including the recalcitrant exon 20 mutations. IAM1363, discovered through Iambic Therapeutics' AI-driven discovery platform, represents the only example of a Type II HER2 inhibitor. It was designed to avoid off-target toxicities from EGFR inhibition thus expanding the therapeutic index over existing HER2 inhibitors. In preclinical studies, IAM1363 shows preferential tumor enrichment, a unique feature observed across tumor types and anatomic sites. The promising target specificity and pharmacokinetic profile of IAM1363 offers the potential to bridge gaps left by current standard of care HER2 targeted treatments. Methods: IAM1363-01 is a first-in-human, multicenter, open-label, Phase 1/1b dose escalation and expansion trial evaluating IAM1363 as monotherapy and in combination with trastuzumab. The study will enroll patients with relapsed/refractory malignancies that have documented HER2 gene alterations (amplification or tyrosine kinase domain mutations) or protein overexpression. Prior treatment with HER2 directed therapies is allowed after a washout period. Part 1, dose escalation, will enroll patients with any tumor type. Part 2 will optimize IAM1363 dosing as monotherapy and in combination with trastuzumab. Part 3 consists of a Simon 2-Stage evaluation of IAM1363 as monotherapy in patients with colorectal, non-small cell lung, and bladder cancer with or without brain metastases and in combination with trastuzumab in breast cancer patients with brain metastases. Key objectives are to establish the safety and tolerability of IAM1363, characterize PK/PD, and determine the recommended Phase 2 dose. Exploratory analysis will correlate HER2 expression level/mutation status with tumor response and tolerability. IAM1363 will be given orally once daily at a starting dose of 120 mg/day in up to 4 planned dose levels. Dose escalation employs an accelerated titration design that transitions to a standard 3 + 3 design based on protocol-specific criteria. Up to 287 patients will be enrolled at approximately 20 sites and treated until disease progression or unacceptable toxicity. The trial is designed to establish the safety profile of IAM1363 as a single agent and in combination with trastuzumab and to provide initial proof of antitumor activity in malignancies with HER2 overexpression, gene amplification, or tyrosine kinase mutations, including in patients with brain metastases. Clinical trial information: NCT06253871 .

Covalent-103: A phase 1, open-label, dose-escalation and expansion study of BMF-500, an oral covalent FLT3 inhibitor, in adults with acute leukemia (AL).

TPS6589 Background: FLT3 mutations occur in 25-35% of AML and result from an internal tandem duplication (ITD) of amino acids in the juxtamembrane domain of FLT3 kinase or point mutations in the tyrosine kinase domain (TKD). FLT3-ITD mutations are associated with increased relapse, shorter remission, and decreased disease-free and overall survival. BMF-500 is a novel oral, highly potent and selective covalent inhibitor of FLT3 including wild-type (WT), ITD, TKD, and resistance mutations (e.g., gatekeeper F691). BMF-500 has high affinity for FLT3, lack of cKIT inhibition, and sustained cell-killing despite drug washout (Law et al., ASH 2022 Abst 2756). BMF-500 shows sustained tumor regression and improved survival in subcutaneous and disseminated xenografts of FLT3-m AML. Methods: COVALENT-103 (NCT05918692) is a multicenter, first-in-human study evaluating the safety, tolerability, and antileukemic activity of escalating doses of daily BMF-500 in patients with relapsed or refractory (R/R) AL, including AML, ALL, or MPAL, with or without FLT3-m. The trial has 2 arms that dose escalate in parallel: Arm A (without) and Arm B (with) concomitant use of a CYP3A4 inhibitor. Using an accelerated titration design (ATD), doses of BMF-500 are escalated in single-subject cohorts until one subject experiences ≥Grade 2 related adverse event or dose-limiting toxicity (DLT). At that point, the cohort will switch to a classical “3 +3” design. Patients with WT FLT3 AL may enroll up to 33% per arm. Treatment continues in 28-day cycles until progression or intolerability. Expansion cohorts will enroll additional patients to obtain further safety and efficacy data. Patients must be refractory, relapsed or have progressed on or following discontinuation of the most recent anti-cancer therapy or be ineligible for any approved standard of care, including hematopoietic stem cell transplant (HSCT). FLT3-positive AML patients must have received a FLT3 inhibitor approved for R/R FLT3-m AML. Additional inclusion criteria include ECOG PS ≤2, adequate organ function, and documented FLT3 mutation status. Key exclusion criteria include known CNS disease, clinically significant cardiovascular disease, and WBC >50,000/µL (uncontrollable with cytoreductive therapy). The primary objective is to evaluate safety and tolerability and determine the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-500 monotherapy based on available pharmacokinetic/pharmacodynamic (PK/PD), safety and efficacy data. Secondary objectives include characterization of the PK/PD of BMF-500, and assessment of its antitumor activity per modified Cheson (2003) criteria or NCCN Clinical Practice Guidelines (ALL Version 1.2022) as determined by the investigator. The study was initiated in July 2023, is currently in dose escalation, and we plan to enroll approximately 110 patients total. Clinical trial information: NCT05918692 .

Beamion LUNG-2: A phase III randomized controlled trial of zongertinib (BI 1810631) versus standard of care (SoC) in patients with locally advanced/metastatic non-squamous non-small cell lung cancer (NSCLC) harboring HER2 tyrosine kinase domain (TKD) mutations.

TPS8654 Background: HER2mutations are present in 2–4% of NSCLC tumors, and ~50% occur in the TKD. First-line SoC for patients with HER2mutation-positive (m+) NSCLC is platinum-based chemotherapy ± immunotherapy; however, no targeted first-line treatment has been approved. Zongertinib is a HER2-selective tyrosine kinase inhibitor (TKI) that binds to both wild-type and mutated HER2 but spares EGFR. In a Phase Ia trial in 53 pre-treated patients with HER2 aberration-positive solid tumors, zongertinib conferred an objective response (OR)/disease control rate (DCR) of 49/91% and an OR/DCR of 58/97% in those patients with HER2m+ NSCLC (n=36), with manageable safety with few EGFR-associated toxicities (Beamion LUNG-1; NCT04886804). Here, we describe Beamion LUNG-2 (NCT06151574), a Phase III, randomized, active-controlled, open-label trial designed to assess the efficacy of first-line zongertinib versus SoC in patients with HER2m+, locally advanced or metastatic non-squamous NSCLC. Methods: Approximately 270 patients from ~160 sites across 30 countries will be randomized 1:1 to receive either zongertinib or SoC and stratified by the presence of the A775_G77insYVMA HER2 mutation. In the experimental treatment arm, patients will receive 120 mg oral zongertinib once-daily in 21-day cycles. In the comparator treatment arm, patients will receive 500 mg/m2 intravenous pemetrexed chemotherapy, plus either 75 mg/m2 cisplatin or Area Under the Curve 5 carboplatin, plus 200 mg pembrolizumab on Day 1 once every 3 weeks (q3w) for four 21-day cycles, then maintenance with 500 mg/m2 pemetrexed plus 200 mg pembrolizumab q3w for up to 35 cycles. Treatment in both arms will continue until progressive disease (PD; RECIST 1.1), undue toxicity, or other criteria are met. The primary endpoint is progression-free survival (RECIST 1.1), by blinded central independent review. Secondary endpoints include OR (defined as best overall response of complete or partial response, RECIST 1.1); patient-reported outcomes analysed as changes from baseline to Week 25; overall survival and occurrence of adverse events (CTCAE version 5.0). Key inclusion criteria are histologically or cytologically diagnosed advanced and/or metastatic non-squamous NSCLC; ≥1 measurable lesion (RECIST 1.1); no prior systemic treatment for locally advanced or metastatic disease; a documented HER2 TKD mutation; and eligibility to receive the selected SoC. Key exclusion criteria are tumors with targetable alterations with an available treatment; presence/history of uncontrolled/symptomatic leptomeningeal disease; and radiotherapy or major surgery ≤4 weeks prior to randomization. Clinical trial information: NCT06151574 .

A Retrospective Analysis of BCR-ABL-1 Kinase Domain Mutations in Frontline TKI Resistant Chronic Myeloid Leukemia Patients: A Single Centre Experience.

CML is a commonly diagnosedmyeloproliferative neoplasm in India. Tyrosine kinase inhibitors (TKIs) are the current standard of care for management of CML. Mutations in Tyrosine kinase Domain (TKD) result inresistance to TKIs. The aim ofthis study isto evaluate the pattern of TKD mutations in CML patients having inadequate response or resistance to first line TKIs and to analyse the outcome of CML patients with and without mutations. It is a retrospective observational study. Medical records of 1633 CML patients from year 2014 to 2021 were analysed. Out of these 108 patients (6.6%) lost their response or did not achieve it in defined time points. 62 patients (71%) were found to have TKD mutations. On analysing specific mutations T315I was the most common mutation seen in 29 (46%) cases followed by M351T in 10 (16%) cases and G250E in 7 (11%) cases. ATP binding region was found to be the most common site in tyrosine kinase domain (50% cases) followed by P-loop (22%) and A loop (9.6%). 13 (20%) cases had ≥2 TKD mutations. The study showed inferior overall survival (OS) in patients with TKD mutations involving T315I mutations and ATP binding domain. Patients may have have underlying TKD mutations at prestation or may develop during course of disease. In case of TKI resistance, testing for specific mutations must be done and appropriate TKI sensitive to underlying mutation is to be used which translates into improved OS.

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. 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. Significance: The identification of targetable genomic subsets of cancer has revolutionized precision oncology and offers patients treatments with more selective and effective agents. Here, we demonstrate that activating, oncogenic MET tyrosine kinase domain mutations are found across a diversity of cancer types and are responsive to MET tyrosine kinase inhibitors.

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.

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

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

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.