Point Mutations In Kinase Domain Research Articles

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 .

Current knowledge about FLT3 gene mutations, exploring the isoforms, and protein importance in AML.

Acute myeloid leukaemia (AML) is a complex haematological malignancy characterised by diverse genetic alterations leading to abnormal proliferation of myeloid precursor cells. One of the most significant genetic alterations in AML involves mutations in the FLT3 gene, which plays a critical role in haematopoiesis and haematopoietic homeostasis. This review explores the current understanding of FLT3 gene mutations and isoforms and the importance of the FLT3 protein in AML. FLT3 mutations, including internal tandem duplications (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD), occur in 25-30% in AML and are associated with poor prognosis. FLT3-ITD mutations lead to constitutive activation of the FLT3 signalling pathway, promoting cell survival and proliferation. FLT3-TKD mutations affect the tyrosine kinase domain and affect AML prognosis in various ways. Furthermore, FLT3 isoforms, including shorter variants, contribute to the complexity of FLT3 biology. Additionally, nonpathological polymorphisms in FLT3 are being explored for their potential impact on AML prognosis and treatment response. This review also discusses the development of molecular treatments targeting FLT3, including first-generation and next-generation tyrosine kinase inhibitors, highlighting the challenges of resistance that often arise during therapy. The final chapter describes FLT3 protein domain rearrangements and their relevance to AML pathogenesis.

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.

Covalent-103: A Phase 1, Open-Label, Dose-Escalation, and Dose-Expansion Study of Bmf-500, an Oral Covalent FLT3 Inhibitor, in Adults with Acute Leukemia (AL)

Background: FLT3 mutations occur in 25-35% of patients with AML and are associated with poor prognosis. FLT3 mutations are most frequently the result of an internal tandem duplication (ITD) of amino acids in the juxtamembrane region of FLT3 or point mutations in the tyrosine kinase domain (TKD). FLT3-ITD mutations are associated with increased incidence of relapse, shorter duration of remission, and decreased disease-free and overall survival. BMF-500 is a novel orally bioavailable, highly potent and selective covalent inhibitor of FLT3 including wildtype (WT), ITD, TKD, as well as a variety of additional resistance-conferring mutations such as the gatekeeper F691. BMF-500 has demonstrated high affinity for FLT3, lack of cKIT inhibition, and sustained cell-killing capacity despite drug washout (Law et al., ASH 2022 Abstract 2756). BMF-500 has shown sustained tumor regression and improved survival in both subcutaneous and disseminated xenograft models of mutant FLT3-driven AML. Study Design: COVALENT-103 (NCT05918692) is an open-label, non-randomized, multicenter, first-in-human Phase I study evaluating the safety, tolerability, and clinical activity of escalating doses of twice daily oral BMF-500 in patients with relapsed or refractory (R/R) AL, including AML, ALL, or MPAL, with or without FLT3 mutations. The trial has 2 arms that will undergo dose escalation in parallel: Arm A (without) and Arm B (with) concomitant use of a moderate or strong CYP3A4 inhibitor. Utilizing an accelerated titration design (ATD), doses of BMF-500 will be escalated in single-subject cohorts until 1 subject experiences either a Grade 2 or higher 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 be enrolled, up to a limit of 33% per arm. Treatment will continue 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 must have progressed on or following discontinuation of the most recent anti-cancer therapy or be ineligible for any approved standard of care therapies, including HSCT. Participants with FLT3-mutant AML must have received treatment with a FLT3 inhibitor approved for treatment of relapsed or refractory FLT3-mutant AML. Key inclusion criteria include ECOG PS ≤ 2, adequate organ function, and documented FLT3 mutation status. Key exclusion criteria include known CNS disease involvement, clinically significant cardiovascular disease, and WBC count >50,000/µL (uncontrollable with cytoreductive therapy). Objectives: The primary objective of the study is to evaluate safety and tolerability and to determine the optimal biological dose (OBD)/ recommended Phase 2 dose (RP2D) of BMF-500 oral monotherapy based on evaluation of available PK/ PD, safety and efficacy data. Secondary objectives include characterization of the pharmacodynamics and pharmacokinetics of BMF-500, and assessment of its antitumor activity per modified Cheson (2003) criteria or the NCCN Clinical Practice Guidelines (ALL Version 1.2022) as determined by the investigator. Endpoints include best overall response rate (ORR), complete remission (CRc), duration of response (DOR), relapse-free survival (RFS) and overall survival (OS). The study was initiated in July 2023 and will enroll ~110 participants at approximately 30 sites.

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.

BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

sImatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but the primary and acquired imatinib resistance remains the big hurdle. Molecular mechanisms for CML resistance to tyrosine kinase inhibitors, beyond point mutations in BCR-ABL kinase domain, still need to be addressed. Here, we demonstrated that thioredoxin-interacting protein (TXNIP) is a novel BCR-ABL target gene. Suppression of TXNIP was responsible for BCR-ABL triggered glucose metabolic reprogramming and mitochondrial homeostasis. Mechanistically, Miz-1/P300 complex transactivates TXNIP through the recognition of TXNIP core promoter region, responding to the c-Myc suppression by either imatinib or BCR-ABL knockdown. TXNIP restoration sensitizes CML cells to imatinib treatment and compromises imatinib resistant CML cell survival, predominantly through the blockage of both glycolysis and glucose oxidation which results in the mitochondrial dysfunction and ATP production. In particular, TXNIP suppresses expressions of the key glycolytic enzyme, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), potentially through Fbw7-dependent c-Myc degradation. In accordance, BCR-ABL suppression of TXNIP provided a novel survival pathway for the transformation of mouse bone marrow cells. Knockout of TXNIP accelerated BCR-ABL transformation, whereas TXNIP overexpression suppressed this transformation. Combination of drug inducing TXNIP expression with imatinib synergistically kills CML cells from patients and further extends the survival of CML mice. Thus, the activation of TXNIP represents an effective strategy for CML treatment to overcome resistance.

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.

Next treatment for TKI-resistant CML

Tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML) in the chronic phase. However, only 50-60% of patients stay on the same TKI for 5 years, and the long-term progression-free survival rate is significantly reduced if an early molecular response is not achieved. Possible mechanisms of therapeutic resistance against BCR::ABL1 dependent clones include point mutations in the ABL1 kinase domain, BCR::ABL1 splicing variants, BCR::ABL1 overexpression, and altered pharmacokinetics by the ABC transporter. Ponatinib, the most potent inhibitor among TKIs, and the STAMP inhibitor asciminib are important drugs for overcoming BCR::ABL1-dependent resistance.

Outcomes in Patients with FLT3-Mutated Relapsed/ Refractory Acute Myelogenous Leukemia Who Underwent Transplantation in the Phase 3 ADMIRAL Trial of Gilteritinib versus Salvage Chemotherapy

The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib improved the survival of patients with relapsed or refractory (R/R) FLT3-mutated acute myelogenous leukemia (AML) in the phase 3 ADMIRAL trial. In this study, we assessed survival and relapse rates of patients in the ADMIRAL trial who underwent hematopoietic stem cell transplantation (HSCT), as well as safety outcomes in patients who received post-transplantation gilteritinib maintenance therapy. ADMIRAL was a global phase 3 randomized controlled trial that enrolled adult patients with FLT3-mutated R/R AML. Patients with R/R AML who harbored FLT3 internal tandem duplication mutations in the juxtamembrane domain or D835/I836 point mutations in the tyrosine kinase domain were randomized (2:1) to gilteritinib (120 mg/day) or to preselected high- or low-intensity salvage chemotherapy (1 or 2 cycles). Patients in the gilteritinib arm who proceeded to HSCT could receive post-transplantation gilteritinib maintenance therapy if they were within 30 to 90 days post-transplantation and had achieved composite complete remission (CRc) with successful engraftment and no post-transplantation complications. Adverse events (AEs) during HSCT were recorded in the gilteritinib arm only. Survival outcomes and the cumulative incidence of relapse were assessed in patients who underwent HSCT during the trial. Treatment-emergent AEs were evaluated in patients who restarted gilteritinib as post-transplantation maintenance therapy. Patients in the gilteritinib arm underwent HSCT more frequently than those in the chemotherapy arm (26% [n = 64] versus 15% [n = 19]). For all transplantation recipients, 12- and 24-month overall survival (OS) rates were 68% and 47%, respectively. Despite a trend toward longer OS after pretransplantation CRc, post-transplantation survival was comparable in the 2 arms. Patients who resumed gilteritinib after HSCT had a low relapse rate after pretransplantation CRc (20%) or CR (0%). The most common AEs observed with post-transplantation gilteritinib therapy were increased alanine aminotransferase level (45%), pyrexia (43%), and diarrhea (40%); grade ≥3 AEs were related primarily to myelosuppression. The incidences of grade ≥III acute graft-versus-host disease and related mortality were low. Post-transplantation survival was similar across the 2 study arms in the ADMIRAL trial, but higher remission rates with gilteritinib facilitated receipt of HSCT. Gilteritinib as post-transplantation maintenance therapy had a stable safety and tolerability profile and was associated with low relapse rates. Taken together, these data support a preference for bridging therapy with gilteritinib over chemotherapy in transplantation-eligible patients.

Olverembatinib in chronic myeloid leukemia.

The introduction of tyrosine kinase inhibitors (TKIs) represents a new era in the management of chronic myeloid leukemia (CML). Despite their long clinical success, point mutations emerging before or during TKI treatment remain an obstacle for several cases. T315I is one of these point mutations in the tyrosine kinase domain of BCR::ABL1. It is a major cause of resistance against first- and second-generation TKIs and therefore lowers survival rates of a small group of patients. Olverembatinib (HQP-1351, formerly GZD-824) is a novel, orally active TKI, which acts through targeting the ATP-binding site of the BCR::ABL1 tyrosine kinase. In recent studies, olverembatinib appears to be an effective and safe treatment option for CML patients harboring T315I mutation. This article mainly focuses on the efficacy and safety data of olverembatinib along with other clinically available and potentially active drugs against T315I-mutated CML.

AML-165 Clinical Outcomes of Patients With FLT3-Mutated Acute Myeloid Leukemia (AML): A Single-Center Retrospective Review

Fms-like tyrosine kinase-3 (FLT3) gene activating mutations predominate in AML, with internal tandem duplication (ITD) in 25 to 35% and point mutations in 5 to 7% of patients with normal karyotype. They associate with shorter relapse-free survival (RFS) and inferior outcome. To examine targeted FLT-3 mutations therapy during induction chemotherapy (IC) of AML. We reviewed all FLT3 mutated AML (both ITD and point mutations in the tyrosine kinase domain (TKD)) patients treated with FLT-3 targeted therapy during IC from March 2013 till December 2021. 28 patients (de novo AML=22, APL=6) with FLT3 mutated AML were identified. Fit patients received IC with fludarabine, cytarabine, and idarubicin (FIA) or IA (3+7 regimen). Older patients received hypomethylating agents (HMA) alone or with venetoclax (Ven). All received IC with FLT3 inhibitors (FLT3I, gilteritinib). All 6 APL patients achieved CR with arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA). Analysis included overall response rate (ORR), complete response (CR), and relapse-free survival (RFS). 14 patients (50%) were men. Races included Whites (n=9), Blacks (n=6), Hispanics (n=8) and Others (n=5). The median age was 54 years (range, 20-68). 20 (71.40%) had ITD mutation, 4 (14.20%) had TKD mutation and 4 had both. 15 had NPM1 and 3 DNMT3A mutations. 14 patients had intermediate and 8 adverse risk AML (ELN). Of the AML patients 14 received standard, IC (FIA=8, IA=2, FIA+FLT3I=4), and 12 (85%) achieved CR. Unfit patients received (HMA alone=2, HMA+ Ven=4, HMA+Ven+FLT3I=1, HMA+FLT3I=1). The ORR was 60% with 50% CR. One FIA dead during IC. Three AML patients (10.70%) had allogeneic stem cell transplantations (ASCTs) and had remained in CR. Two non-ASCT were treated with HMA and one with FLT3I maintenance remaining in CR. 4 patients (14.20%) died in remission. Median relapse-free survival (RFS) was 360 days (range, 218-669). The median RFS for non-ASCT patients was 240 days (range, 195-405). Historically RFS in FLT-3 AML is 120 days. IC with FLT3I is highly effective with low induction mortality. Response duration was shorter in the non-ASCT group. In FLT-3 AML ASCT ineligible patients, maintenance therapy may reduce relapse risk.

P461: MYELOID KINOME INHIBITOR HM43239 OVERCOMES ACQUIRED RESISTANCE IN ACUTE MYELOID LEUKEMIA MODELS

Background: Oral HM43239 is in development for the treatment of acute myeloid leukemia (AML) because of its capacity to potently inhibit kinases that drive myeloid malignancies, including diverse forms of the FLT3, SYK, JAK and c-KIT kinases. Wildtype FLT3 is overexpressed in most AML patients, and approximately 30% of newly diagnosed adult AML patients harbor internal tandem duplications (ITDs) or point mutations in the tyrosine kinase domain (TKD). These mutations drive aberrant activation of downstream proliferation pathways and are associated with a high risk of relapse. Likewise, the c-KIT alternative receptor kinase, as well as the SYK and JAK1/2 intracellular kinases, mediate oncogenic signaling in AML that can promote drug resistance to certain FLT3 inhibitors. HM43239 was developed to overcome shortcomings of other FLT3 inhibitors. It inhibits a broad set of mutant and wildtype forms of FLT3, while simultaneously disrupting downstream SYK, JAK/STAT5, ERK, and other rescue signaling pathways. This rationale supports the development of HM43239. Aims: Evaluate the activity of HM43239, an orally active drug, as a FLT3-focused myeloid kinome inhibitor in human AML models. Methods: Biochemical kinase assays were performed by RBC and Carna. The effects of HM43239 on cell proliferation (IC50), growth rate (GR50) and concentration at half-maximal effect (Growth Effective Concentration; GEC50) were determined using the MTS assay with vehicle controls. Cell-based inhibition of target phosphorylation was assessed by Western blot and flow cytometric analyses. The AML cell lines tested included MV-4-11, MOLM-13, MOLM-14 and BAF3/ITD. In vivo efficacy was assessed using the MOLM-14 FLT3-Mutated xenograft model. Results: HM43239 inhibited the enzymatic activities of FLT3-WT, -ITD, and -D835Y variants with IC50 values of 1.1, 1.8 and 1.0 nM, respectively. Binding Kd values for FLT3-WT, -ITD, -D835Y, -D835H, -ITD/D835V and -ITD/F691L were 0.58, 0.37, 0.29, 0.4, 0.48, and 1.3 nM, respectively. HM43239 killed AML cells that harbor the FLT3-ITD mutation (MV-4-11, MOLM-13 and MOLM 14) with IC50, GR50 and GEC50 values ranging from 4 to 10 nM. In cell lines expressing wild type FLT3 (KG1, HEL92.1, SKM-1, and THP-1) the values for these parameters ranged from 0.05 nM to 3 µM. Western blot and flow cytometric analyses of MV-4-11, MOLM-14 cells and Ba/F cells transfected with target proteins revealed that the phosphorylation of FLT3 and SYK was reduced by 50-90% at HM43239 concentrations of 10 – 100 nM, and that these concentrations produced >80% reduction in phosphorylation of ERK and JAK/STAT5 that function downstream of FLT3. HM43239 inhibited FcγR-induced SYK and JAK/STAT5 activation in KG-1a (FLT3-WT) cells that upregulate RAS signaling which is a mechanism of acquired resistance to gilteritinib. In murine xenograft studies, HM43239 was more effective than gilteritinib in both SC and orthotopic FLT3-F691L and FLT3-ITD/F691L mutant MOLM-14 models. Summary/Conclusion: HM43239 inhibits wild type and mutant forms of FLT3 at low nM concentrations and demonstrates in vivo efficacy at doses that are well tolerated. Its ability to also inhibit SYK and, by reducing the activity of these upstream kinases, to also impair the activity of EKR1/2 and JAK/STAT5 that participate in rescue pathways, makes this a particularly interesting molecule with the potential of offsetting the development of resistance that is common with other FLT3 inhibitors. A Phase 1/2 trial of HM43239 in patients with AML is open and accruing patients (NCT03850574).

Abstract 5456: Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations

Abstract FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5456.

A Phase 1a/1b First in Human Study of PHI-101, a Potent Small Molecule Inhibitor of FLT3 in Relapsed and Refractory Acute Myeloid Leukemia

Background: The FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients either by internal tandem duplication (ITD) or by point mutations in the tyrosine kinase domain (TKD). PHI-101 is a Type I FLT3 inhibitor developed to overcome resistance in AML. PHI-101 showed potent cellular activity in vitro and in vivo for both FLT3 ITD mutations and a wide variety of FLT3 point mutations in TKD. In preclinical studies utilizing primary human FLT3-ITD leukemia cells, PHI-101 showed increased anti-leukemic activity compared to gilteritinib based on increased survival in mice transplanted with these samples.The Phase 1a and Phase 1b global clinical trial with PHI-101 (NCT04842370) is currently underway to assess the overall safety and efficacy of PHI-101 in refractory and relapsed AML.Study Design and Methods: The overall design of the AML clinical trial consists of two parts, phase 1a, the dose-escalation, and phase 1b, the dose-expansion trial utilizing oral PHI-101 tablets. Up to 5 dose levels are planned for phase 1a and a single subject enrolled per dose level until the occurrence of at least one subject with more than Grade 2 toxicity according to CTCAE5.0 criteria during the 28-day evaluation period. The dose escalation will then convert to a standard 3+3 scheme, with 3 to 6 subjects per dose level cohort over the 28-day cycle. If there are no dose-limiting toxicities (DLT), dose escalation to the next higher dose levels will proceed upon the recommendation of the Safety Monitoring Committee. Subjects with FLT3 mutations or FLT3 wild-type will be enrolled in the dose-escalation cohort, and blood samples are collected for the primary endpoint pharmacokinetics (PK) and for the exploratory pharmacodynamic (PD) endpoint evaluation, including plasma inhibitory assay and biomarker analysis.Results: The Phase 1a clinical trial of PHI-101 was initiated in June 2020 at a daily dose of 40mg for level 1. As of July 31, 2021, 8 patients with relapsed or refractory AML have been enrolled in the trial, all of whom had prior anti-leukemic treatments with intensive chemotherapy, hypomethylating agents and/or other FLT3 inhibitors. To date, 5 enrolled patients were available for safety assessment at three dose levels and have not experienced any dose-limiting toxicities (DLTs) from repeating doses of the 28-day DLT-assessment window. The leukemic blasts in marrow or peripheral blood were dramatically reduced by up to 98% with one cycle (28 days). The pharmacokinetic data of plasma PHI-101 varied in a dose-proportional manner and peak plasma concentrations (Cmax) were reached between 4 and 6 hours after once-daily dosing. The pharmacodynamic evaluation by plasma inhibitory assay showed that plasma from patients receiving at daily doses of PHI-101 inhibited more than 90% of the phosphorylation of the FLT3-ITD receptor. PHI-101 was well tolerated with these dose levels. This study is currently recruiting FLT3 mutation or wild-type AML patients at multiple sites in Korea and Australia.Conclusion: PHI-101 is a next-generation FLT3 inhibitor that showed a potent anti-leukemic activity and improved efficacy in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations in preclinical studies. The current analysis of this trial indicated that PHI-101 is a very effective FLT3 inhibitor for both refractory and relapsed AML patients, including those that have relapsed on other FLT3 TKI. The assessments of safety, tolerability, and PK of PHI-101 to determine the recommended dose for expansion are ongoing in the Phase 1a clinical trial. DisclosuresLee: Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board; Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees. Im: Pharos iBio Co., Ltd.: Current Employment. Nam: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Kim: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Han: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Yoon: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Small: Pharos iBio Co., Ltd.: Consultancy, Other: Scientific Advisory Board. The arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies, Research Funding; InSilico Medicine: Other: Scientific Advisory Board.

A Systematic Review and Meta-Analysis Comparing Type I and II FLT3 Inhibitors in Relapsed/ Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome

Background: Acute myeloid leukemia (AML) is frequently characterized by mutations in FMS-like tyrosine kinase 3 (FLT3). FLT3 is mutated in one-third of de novo AML patients and recurrent in relapsed/refractory (R/R) AML. More frequent FLT3 internal tandem duplications (ITD) and less frequent tyrosine kinase domain (TKD) point mutations are prognostically important in AML and are associated with an inferior outcome. Although targeted therapy with FLT3 inhibitors improves prognosis in FLT3 mutated AML patients, there is a lack of evidence regarding the superiority of efficacy of various FLT3 inhibitors in clinical practice and the therapeutic pipeline. Broadly grouped as type 1 (targets ITD and TKD, e.g., midostaurin) and type 2 (targets only ITD, e.g., quizartinib) inhibitors, there is no consolidated summary of the difference in efficacy between these two classes of FLT3 inhibitors in R/R AML and high-risk myelodysplastic syndrome (HR-MDS). Hence, we conducted a systematic review and meta-analysis of various type 1 and 2 FLT3 inhibitors tested in clinical trials to compare their clinical effectiveness in treating R/R AML and HR-MDS.Methods: PubMed and EMBASE databases were searched for clinical trials published between 1/1/2000 and 5/26/2021 using a combination of keywords and subject headings related to FLT3 inhibitors and AML. Two independent reviewers screened titles/abstracts and full texts, with a third reviewer resolving conflicts. Studies were included if they were (1) full-length published journal articles that (2) reported the results of single-arm or double-arm phase I/II/III clinical trials in patients with R/R AML or HR-MDS. Outcomes of interest were composite response rate (CRc=complete response + complete response with incomplete count recovery) and overall response rate (ORR).Results: Record screening resulted in the initial inclusion of 30 studies for qualitative analysis (Fig. 1). Due to poor representation of R/R AML patients and post-transplant FLT3 inhibitor outcomes, two studies were excluded from quantitative analysis, leaving 28 studies for the final analysis. The studies had a total of 1927 R/R AML and HR-MDS patients with a median age of 61.5 years (range, 13-91) and a median of three (range, 0-10) prior lines of treatment. Giltertinib (n=3 studies) was the most frequently studied type 1 inhibitor, and quizartinib (n=6) and sorafenib (n=5) were the most frequently studied type 2 inhibitors. Heterogeneity testing using Cochran's Q test and I 2 values showed the presence of heterogeneity for both CRc (p<0.01 and I 2=87% for both type 1 and 2) and ORR (p<0.01 for both type 1 and 2, I 2=88% and 92% for type 1 and type 2, respectively) and. Hence, random-effects models were used. Asymmetry tests performed using Egger's linear regression test suggested publication bias only for type 2 (p=0.005) for ORR and type 1 and 2 for CRc (p=0.017 and 0.002, respectively). Thus, the trim-and-fill method was used to adjust for publication bias for type 2 for ORR and type 1 and 2 for CRc. Pooled ORR was 37% (95% CI, 25-51%) for type 1 and 58% (95% CI, 43-71%) for type 2. The highest ORR was for gilteritinib (68%; 95% CI, 61-73%) among type 1 and sorafenib (99%; 95% CI, 81-100%), linifanib (99%; 95% CI, 89-100%), and tandutinib (100%; 95% CI, 93-100%) for type 2 (Fig. 2). Pooled CRc was 35% (95% CI, 21-52%) for type 1 and 38% (95% CI, 27-50%) for type 2. The highest CRc was for midostaurin (98%; 95% CI, 79-100%) among type 1 and sorafenib (97%; 95% CI, 68-100%), tandutinib (98%; 95% CI, 72-100%), and semaxinib (98%; 78-100%) among type 3 (Fig. 3).Conclusion: Both type 1 and 2 inhibitors are effective as monotherapy in R/R AML and HR-MDS patients. Though not statistically significant, better ORR for type 2 than for type 1 inhibitors in the R/R setting provides a background to explore the biological heterogeneity of response and preferential sensitivity to ITD-targeted therapy alone rather than the dual inhibitor. More prospective randomized study designs comparing type 1 and 2 inhibitors in the R/R and de novo setting are needed. [Display omitted] DisclosuresMaciejewski: Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Novartis: Consultancy. Balasubramanian: Servier Pharmaceuticals: Research Funding.

FDA Cleared Companion Diagnostics (CDx) Tests (IDH1, IDH2 and FLT3) for Acute Myeloid Leukemia (AML) Patient Care

Acute myeloid leukemia (AML) is one of the most lethal blood cancers from which nearly 10,000 people die in the United States each year. While therapies for other blood cancers have made some progress, the standard of care for AML, a combination of toxic chemotherapies, has changed very little over the past four decades. In recent years the US Food and Drug Administration (FDA) has been very active in approving targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations. Additionally, the Leukemia and Lymphoma Society's Beat AML Master Clinical Trial has shown that waiting for molecular results prior to treatment decision leads to better outcomes. Versiti Blood Center of Wisconsin Diagnostics laboratory which is certified under the Clinical Laboratory Improvement Amendments (CLIA) and qualified to perform high complexity clinical laboratory testing has performed the verification studies and offers two companion diagnostics tests for IDH1 and IDH2 mutations for AML patients. Also, in collaboration with Invivoscribe Inc., the AML patient can be tested for Leukostrat CDx FLT3 mutations assay so that the same AML patient can get three CDx test results leading to available drug therapy treatment decision making by physicians.IDH1CDX Test: IDH1 CDx is indicated as an aid in identifying AML patients with an IDH1 mutation for treatment with ivosidenib (TIBSOVO®). Mutations in codon R132 of IDH1 can be found in 6% to 10% of AML patients. The IDH1 CDx test detects five IDH1 mutations R132H (CAT), R132C (TGT), R132G (GGT), R132S (AGT), and R132L (CTT) using PCR technology with homogeneous real-time fluorescence detection. The assay sensitivity for these five IDH1 mutations is 100% at variant allele frequencies of 2% and higher and 98% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170041).IDH2CDx: IDH2 CDx is indicated as an aid in identifying AML patients with an IDH2 mutation for treatment with IDHIFA® (enasidenib). Mutations in the R140 and R172 codons of IDH2 8% to 19% of AML patients.The IDH2 CDX test detects nine IDH2 mutations (R140Q, R140L, R140G, R140W, R172K, R172M, R172G, R172S, and R172W) using PCR technology with real-time fluorescent detection. The assay sensitivity for these nine IDH2 mutations is 99.8% or greater at variant allele frequencies of 2% and higher or 93.5% or greater at variant allele frequencies of 1% and higher. This test has been approved by the FDA as companion diagnostic device (PMA number P170005).FLT3CDx: The FLT3 Leukostrat® CDx Assay is the FDA approved (PMA number P160040) predictive test for the efficacy of midostaurin (RYDAPT®) therapy in all AML patients, regardless of cytogenetics and efficacy of gilteritinib (XOSPATA ® ) therapy in relapsed or refractory AML patients. FLT3 is one of the most commonly mutated genes in AML with 30% of patients at the time of diagnosis 1. The most prevalent type of FLT3 mutation is an internal tandem duplication (ITD) in the juxtamembrane domain. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in the codon for an aspartate (D835) or an isoleucine (I836) residue. The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations and the tyrosine kinase domain (TKD) mutations D835 and I836 in genomic DNA extracted from mononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with AML. Versiti Blood Center sends the patient specimens to Invivoscribe Inc. where the LeukoStrat® CDx FLT3 Mutation Assay is performed and the interpretive comments are included in the patient report by Versiti.From our experience pathologists and treating physicians want molecular test results as fast as possible, especially for the actionable gene mutations in IDH1, IDH2 and FLT3. The IDH1 CDx, IDH2 CDx and FLT3 CDx tests are highly sensitive and Versiti provides average turn around time of 3 business days which enable rapid decision making on the recently available drug therapies for AML patients. We strongly recommend that the IDH1 CDx, IDH2 CDx and FLT3 CDx tests should be performed on all AML patients for better care. DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:In recent years the US Food and Drug Administration (FDA) approved targeted therapeutic drugs for AML patients including Midostaurin (Rydapt; 2017) and Gilteritinib (Xospata;2018) for FLT3 mutations; Enasidenib (Idhifa; 2017) for IDH2 mutations and Ivosidenib (Tibsovo; 2018) for IDH1 mutations.

FLT3-Mutated Acute Myeloid Leukemia Using a Novel Regimen of Gemtuzumab Ozogamicin and Midostaurin in Combination with Standard Cytarabine and Daunorubicin Induction Therapy

FLT3-Mutated Acute Myeloid Leukemia Using a Novel Regimen of Gemtuzumab Ozogamicin and Midostaurin in Combination with Standard Cytarabine and Daunorubicin Induction Therapy

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

AML-347: A Preliminary Analysis of FLAM (Italian Non-Interventional Multi-Center Study of FLT3-Mutated AML Patients): FLT3 Receptor Gene Mutational Analysis and FLT3 Inhibitor Administration in Real-Life Clinical Practice

Oncogene-independent resistance in Philadelphia chromosome - positive (Ph+) acute lymphoblastic leukemia (ALL) is mediated by activation of AKT/mTOR pathway.

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells.Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance.

Spectrum of BCR-ABL Mutations and Treatment Outcomes in Ethiopian Imatinib-Resistant Patients With Chronic Myeloid Leukemia.

PURPOSEDespite the successes achieved in chronic myeloid leukemia (CML) with tyrosine kinase inhibitor (TKI) therapy, resistance remains an obstacle. The most common mechanism of resistance is the acquisition of a point mutation in the BCR-ABL kinase domain. Few studies have reported African patients with CML in regard to such mutations. We here report the types of BCR-ABL mutations in Ethiopian imatinib-resistant patients with CML and their outcome.PATIENTS AND METHODSPatients with CML with a diagnosis of imatinib resistance who were tested for BCR-ABL mutation between 2014 and September 2019 were included.RESULTSA total of 962 cases of CML on imatinib therapy were reviewed and 164 cases of failure were found. Of these, only 31 cases (19%) had mutation analysis performed. Most cases (94%) were secondary failures. At the time of CML diagnosis, the median age was 33 years and the majority presented with features of advanced-phase disease. Of the 31 patients, 22 mutations were found (65%). The types of mutations detected were as follows: non–P-loop mutations 36% (11), P-loop mutations 13% (four), and alternatively spliced BCR-ABL variants 23% (seven). The splice variant frequently detected was BCR-ABL35INS (20%). Twenty-six of the 31 patients (84%) were switched to second-line TKIs, whereas in four patients (13%), imatinib dose escalation was done. Overall, the outcome revealed that 16 patients (52%) were alive with complete hematologic response, whereas 12 patients (39%) had died. All patients who expressed BCR-ABL135INS were treated with second-line TKIs, and two of them (33%) had died because of disease progression.CONCLUSIONIn Ethiopia, CML affects the young and point mutations were frequently detected in imatinib-resistant patients. BCR-ABL1 35INS was also prevalent and associated with disease progression.