F317L Mutation Research Articles

Safety and Efficacy of Flumatinib in Patients with Chronic Myeloid Leukemia Resistant or Intolerant to Imatinib

INTRODUCTION:Flumatinib mesylate is a derivative of imatinib and has higher selectivity and potency toward BCR::ABL1 kinase compared with imatinib. Flumatinib was approved patients with newly diagnosed CML-CP in China. We analysed the efficiency and safety of flumatinib in CML patients resistant or intolerant to Imatinib in the real word. METHODS:164 adult CML-CP patientsrecieved flumatinib as 2nd line therapy after imatinib were collected from 18 centers in china. The primary outcome was to demonstrate the probabilities of responses including complete hematologic response (CHR), cytogenetic response, and molecular response (MR) after the later line of flumatinib. The secondary outcome was to assess adverse events (AEs). The diagnosis and response evaluation were defined according to the European Leukemian Net 2020 recommendations. The side effect were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.0. Results: 164 patients with CML-CP with a median age of 51 (range: 17-87) years were included, 95(57.9%) males. The medium interval from imatinib to flumatinib was 13.2(1-185) months. The median dosage of flumatinib was 600 mg/day. 127 patients had ELTS information with 84 (66.1%), 30 (23.6%), 13 (10.2%) patients in low-, medium-, and high risk groups, respectively. ABL mutations were detected in 144 patients before initiating flumatinib, 5 (3.5%) patients had ABL mutations with 1 patient each showing E279K, E459K, M351T mutation; and 2 patients had F317L mutation. I 56 (34.1%) patients had optimal response but intolerant to imatinib, whereas 43 (26.2%), 65 (39.5%) patients had suboptimal or failure response to imatinib. The median duration of flumatinib treatment was 10.9 (2-25.46) months. Discontinuation of flumatinib treatment was observed in 17 (11%) patients, with a median treatment duration of 8.8 (2-25.46) months. The probabilities of CHR, CCyR/MR 2, MMR, and MR4 or better at baseline and after flumatinib therapy have been represented in Table 1. The patients without evaluable data were considered without response. The rate of CHR, CCyR/MR 2, MMR and MR4 or better were 86% (141/164), 48.2% (79/164), 15.2% (25/164) and 7.9% (13/164) at baseline increased to 97.6% (160/164). 81.1% (133/164), 65.2% (107/164), 34.8% (57/164) respectively during flumatinib treatment. The probabilities of response from CHR to MR after flumatinib therapy were analyzed based on clinical parameters. The gender, age, and ELTS score at diagnosis had no influence on the response to flumatinib, whereas the response to imatinib, the interval from imatinib to flumatinib, transcript levels at baseline were associated with the response to flumatinib. Patients who were intolerant to imatinib had higher rates achieving CCyR/MR 2, MMR, and DMR when compared with those with warning or failure response to imatinib. The median time to CCyR/MR 2, MMR, and DMR was 3.7 months, 4.7 months, and 15 months in intolerant patients; 3.8 months, 7.5 months, and not reached in warning patients; 7.1 months, 8.7 months, and not reached in failure patients, respectively. The patients with transcript ≤10% at baseline showed a higher probability of achieving CCyR/MR 2 and MMR as compared with those with transcript >10% at baseline, while the probabilities to achieve DMR were similar in both groups. The patients switched to flumatinib within 6 months and without CCyR/MR2 at baseline had a higher probability and shorter medium time of achieving CCyR (85.3% vs 51%, 3.3 vs 9.9months). The patient with E279Km and resistant to imatinib achieved MMR with flumatinib treatment. 1 patient with F317Lm achieved MR4. The patient with M351Tm received flumatinib for 4.5 months and with decreasing transcript level, 1 patient with E459Km received flumatinib for 4 months and maintained CHR with stable BCR::ABL transcript levels. 27 patients had hematological toxicities during exposure to Flumatinib. Most nonhematological AEs were grade ≤2, and only 2 patients showed grade ≥3 diarrhea. The major flumatinib-related nonhematological AEs included gastrointestinal AEs ( n = 29), rash ( n = 9), and creatinine elevation ( n =7). conclusion：This retrospective study had suggested promising effects of flumatinib, which showed induced high rates of CCyR and MMR or DMR in patients resistant or intolerant to imatinib. The incidence of AEs during flumatinib treatment was tolerable.

Therapeutic Targeting of FLT3 Gate Keeper Mutation with E2082-0047 in Traditional and a Novel Immunocompetent Murine Adoptive Transfer Model of AML

Signaling mutations such as Fms-like tyrosine kinase 3 (FLT3) internal tandem duplication (FLT3-ITD) occur in approximately 30% of AML patients which provide AML cells with a constitutively active aberrant growth advantage, resulting in inferior overall survival. Multiple phase 3 studies with different FLT3 inhibitors validated FLT3-ITD as an important therapeutic target in AML. However, resistance to FLT3-ITD inhibitors occurs via mutations in the tyrosine kinase domain (FLT3-TKD), including the gatekeeper mutation F691L for which all approved FLT3 inhibitors are ineffective. Other mechanisms of secondary FLT3 inhibitor resistance include induction of IRAK4 activity and acquisition of RAS/PTPN11/MAPK activating mutations. New FLT3 inhibitors targeting both intrinsic (FLT3 acquired mutations) and extrinsic mechanisms of FLT3 resistance (e.g. IRAK4 induction) are urgently needed. We demonstrated previously the selectivity and potency of a novel FLT3 inhibitor, E2082-0047, against FLT3-ITD and TKD mutations in vitro using a Ba/F3 cell line harboring different TKD mutations. Guided by KinomeScan data, we demonstrated simultaneous IRAK4 inhibition and verified superior in vivo efficacy compared to gilteritinib using the FLT3-ITD MOLM-13 cell line derived xenograft (CDX) murine model (Elgamal OA et al., ASH 2022). Recognizing the exceptional safety profile of E2082-0047 in pre-clinical IND enabling toxicology studies, we moved forward to test the potency of E2082-0047 versus gilteritinib in cells harboring the FLT3-ITD-F691L gatekeeper mutation using two in vivo models, MOLM-14-F691L CDX model and a novel immunocompetent murine adoptive transfer model that does not require conditioning or irradiation. This model is generated by the crossing of the CD45.2 specific Npm1 cA cre inducible model (Vassiliou et al., 2011) with the Flt3 ITD (Lee et al., 2007) model which was previously reported to harbor a F692L mutation, analogous to the human F691L mutation (Dovey et al., 2016) thus, generating the Npm1 cA;Flt3 ITD-F692L AML murine model. In the MOLM-14-F691L CDX model study, we found a consistent reduction in human CD45 cells in bone marrow and increased survival benefit in mice receiving E2082-0047 compared to gilteritinib. Further, mice in the gilteritinib group with progressing disease (> 50% human CD45+ cells in bone marrow) were switched to E2082-0047 with consequent > 5-fold reduction in bone marrow tumor burden within 8 days. These data suggest that E2082-0047 will be effective in gilteritinib resistant disease. Strikingly, in the AML adoptive transfer model, E2082-0047 resulted in a compelling long-term survival benefit. In our first study (donor #1), vehicle and gilteritinib cohorts had 29- and 32-days median survival, respectively with evident circulating CD45.2 AML. In the E2082-0047 cohort, mice remained on study with an average of <2% circulating CD45.2 cells therefore, we stopped treatment and euthanized three mice for tissue collection, leaving eight mice for disease relapse monitoring for an additional two weeks where we lost 3/8 mice due to early removal criteria (ERC) and 5/8 were euthanized at end of study day 100, with an average of <10% circulating CD45.2 cells. This initial study showed the capacity for E2082-0047 to induce long-term survival in a model with a clinically relevant TKD gatekeeper mutation. We repeated our study with a more aggressive donor (donor #2) where vehicle and gilteritinib arms had a median survival of 22 days. While mice receiving E2082-0047 remain on study (week 8 of an ongoing study), with an average of <5% peripheral blood CD45.2 level (Figure 1). Collectively, our previous and follow-up studies herein demonstrate the superiority of E2082-0047 over gilteritinib against FLT3-ITD and with TKD mutations including the gatekeeper F691L variant. We demonstrate significantly improved survival after E2082-0047 treatment in an immune deficient in vivo model of FLT3-ITD/TKD-F691L MOLM-14 AML and a durable long-term survival in a large subset of mice using two donors in an immune competent adoptive transfer model of NPM1+FLT3-ITD/TKD-F692L AML. Given the curative potential of E2082-0047 in the immune competent model, ongoing efforts are focused on investigating the host immune response to AML. Given the safety and efficacy studies performed with E2082-0047 to date, a healthy volunteer study to best estimate dose for trials in AML is ongoing.

Asciminib Enhances Its Treatment Efficacy Synergistically in the Treatment of Chronic Myeloid Leukemia Harboring ABL1 Kinase Domain Mutation When Combined with a Reduced Dose of Ponatinib, Dasatinib, or Bosutinib, but Not with Nilotinib or Imatinib

Background: Asciminib (ASC) monotherapy has been approved for chronic myeloid leukemia (CML) treatment in the patients who failed at least 2 lines of tyrosine kinase inhibitor (TKI) therapy. Theoretically, dual inhibition of the ABL1 kinase protein is plausible by combining ASC with ATP-binding pocket inhibitors (ABPIs) including Imatinib (IMA), Dasatinib (DAS), Nilotinib (NIL), Bosutinib (BOS) or Ponatinib (PON). A phase 1 study determined the appropriate dose of ASC with fixed doses of ABPIs (IMA, DAS, NIL). However, this approach of using full dose ABPIs with variable dose of ASC would raise a concern of tolerability given that all the toxicity from the doses of ABPI will be augmented by adding even half dose of ASC, evidenced by the FASCINATION trial (Ernst et al. EHA 2023). Questions still remained such as what is the best dose for combination? Is the combination of full dose ASC with reduced dose ABPIs effective? Is this combination effective to other ABL1 kinase domain mutant CML? For the ABL1 kinase domain (KD) mutant CML, which ABPI and what dose is more effective and more synergistic than other ABPIs? Methods and Materials: To investigate the combined effect of ASC with ABPIs, we determined the IC 50 values for ASC and ABPIs in the wild-type (WT) BaF3 BCR-ABL+ cell line, and utilized the IC 50 as baselines throughout the study. From the baseline IC 50s of ASC (3nM), IMA (150nM), NIL (10nM), BOS (10nM), DAS (0.8nM) and PON (0.6nM), serially diluted ASC alone, ABPIs alone or in combination were administered to BaF3 cell lines. We conducted IC 50 determining experiment in different combination of ASCs/ABPIs/combination in different concentration (conc) on eleven different KD mutant (KDM) BaF3 cell lines, including WT, G250E, E255K, T315A, M351T, F317L, F317V, H396P, Y253F, M244V, and T315I mutant cell lines. ZIP synergy scores for these cell lines were analyzed using SynergyFinder. Results We observed that the addition of 3 nM ASC to ABPI at baseline IC 50 conc led to a higher response (% cell growth inhibition) compared to treatment with ABPI alone at baseline conc, irrespective of the mutation. Moreover, the combined treatment of ABPI at half baseline conc with 3nM ASC demonstrated similar or even higher effects than ABPI alone at IC 50 baseline conc. These results suggest that double blockade allows for the reduction of ABPI dose by half while maintaining cell inhibition activity. According to its sensitivity to ASC monotherapy and combination, we have stratified the cell lines into 3 categories. High sensitive cell lines to ASC monotherapy (i.e. WT, G250E, and E255K) achieving 100% of response rate when combined with ABPI. Also, T315A mutation showed a significant synergistic effect (ZIP = 12.91~21.75) with the combination of ASC 3nM with a half conc of ABPI, resulting in 1.90-2.43 times higher inhibitory effect than ABPI monotherapy.Intermediate sensitive cell lines (i.e. M351T and F317L) displaying 69-85% inhibition efficiency with ASC 6nM combined with ABPI, without significant increases in all ABPI conc.Low sensitive cell lines (i.e. F317V, H396P, Y253F, M244V, and T315I) exhibiting unique resistance profiles that varied with different combinations. Notably, F317V and F317L mutations showed resistance to DAS. When combined with ASC 3nM, F317V showed restored sensitivity with IMA or NIL than with DAS, while F317L better with DAS. Moreover, the H396P, Y253F, and M244V mutations demonstrated higher sensitivities with BOS or DAS. Interestingly, in the combination treatment with ASC at 3 or 6 nM, with exception of the F317V and T315I mutations, both DAS and PON exhibited a potent efficacy. High resistance of T315I to ABPIs can be overcome with combination of ASC at baseline conc with reduced dose PON. In the case of T315I, the combination of 3nM ASC with a half conc PON resulted in a higher inhibitory effect than baseline conc PON alone. Conclusion: The present study demonstrated that a half of baseline conc ABPIs in combination with ASC is as effective as other ABPIs not just in WT but also in other ABL1 KDM CML cell lines. Different CML cell lines harboring different ABL KDM has different treatment spectrum on optimal ABPI drug as well as optimal drug conc. This result will be useful to design future clinical trial of dual blockade of ASC with other ABPIs considering the ABL KDM profiles. It also emphasizes the importance of ABL KDM testing prior to ASC monotherapy or combination with other ABPIs.

Deciphering the mechanism of HM43239 inhibiting the mutant F691L resistant to gilteritinib in FMS-like tyrosine kinase 3

FMS-like tyrosine kinase (FLT3) has become the legitimate molecular therapeutic target for acute myeloid leukemia therapy. Though FLT3 inhibitors have impact on disease progression, drug resistance induced by secondary point mutations is the primary mechanism and urgent to overcome. Herein, we sought to decipher the mechanism of HM43239 inhibiting the mutant F691L resistant to gilteritinib in FLT3. A series of molecular modeling studies, including molecular dynamics (MD) simulation, dynamic cross-correlation (DCC) analysis, binding free energy (MM-GBSA) and docking study were explored to elucidate the differential tolerance mechanisms of two inhibitors to the same mutant. The F691L mutation had relatively larger effect on gilteritinib than HM43239, which showed as the changed and fixed conformation, respectively. These observations rationalized that the binding affinity of gilteritinib decreased more than that of HM43239 in the F691L mutant. Communicated by Ramaswamy H. Sarma

Genetic alterations in the BCR-ABL1 fusion gene related to imatinib resistance in chronic myeloid leukemia

Use of the potent tyrosine kinase inhibitor imatinib as the first-line treatment in chronic myeloid leukemia (CML) has decreased mortality from 20% to 2%. Approximately 30% of CML patients experience imatinib resistance, however, largely because of point mutations in the kinase domain of the BCR-ABL1 fusion gene. The aim of this study was to use next-generation sequencing (NGS) to identify mutations related to imatinib resistance. The study included 22 patients diagnosed with CML and experiencing no clinical response to imatinib. Total RNA was used for cDNA synthesis, with amplification of a fragment encompassing the BCR-ABL1 kinase domain using a nested-PCR approach. Sanger and NGS were applied to detect genetic alterations. HaplotypeCaller was used for variant calling, and STAR-Fusion software was applied for fusion breakpoint identification. After sequencing analysis, F311I, F317L, and E450K mutations were detected respectively in three different participants, and in another two patients, single nucleotide variants in BCR (rs9608100, rs140506, rs16802) and ABL1 (rs35011138) were detected. Eleven patients carried e14a2 transcripts, nine had e13a2 transcripts, and both transcripts were identified in one patient. One patient had co-expression of e14a2 and e14a8 transcripts. The results identify candidate single nucleotide variants and co-expressed BCR-ABL1 transcripts in cellular resistance to imatinib.

Abstract LB516A: The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms

Abstract Background: FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin and gilteritinib have improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML), but resistance remains a problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 and has shown activity against FLT3 F691L and D835 mutations. We evaluated FF-10101 against an expanded panel of FLT3 resistance mutations and activating mechanisms of MAPK signaling associated with FLT3 TKI resistance in AML cell lines and patient samples. Results: FF-10101 was uniformly active against a series of previously unevaluated FLT3 TKI resistant D835 mutations (sensitivity = IC50 <10X of FLT3-ITD alone in Ba/F3 cells). FF-10101 was more active against F691L than gilteritinib (8-fold vs 14-fold increase in IC50 vs. FLT3-ITD, respectively). K429E and N676K mutations associated with clinical resistance to crenolanib and midostaurin, respectively, were also both FF-10101-sensitive. G697S and Y693N mutations, previously identified by random saturation mutagenesis screening to confer resistance to gilteritinib, also conferred moderate resistance to FF-10101 while Y693C conferred a high degree of resistance to both (IC50 >50X higher than FLT3-ITD). D698N, however, was FF-10101-sensitive and gilteritinib-resistant. Using a cell-based model, we confirmed C695 as the lone residue covalently bound by FF-10101. Moreover, a mutagenesis screen for FF-10101 resistance identified only C695Y/R mutations. Structural modeling showed an aromatic π-π interaction between FF-10101 and Y693 that orients an electrophilic acrylamide portion for covalent bonding of FLT3 at C695, likely explaining resistance conferred by Y693C. Confirmatory mutagenesis experiments showed that Y693F, by recapitulating the native aromatic interaction, retained FF-10101 sensitivity while the non-aromatic Y693S mutation conferred a high degree of resistance. Finally, FF-10101 exerted generally greater cytotoxicity than gilteritinib in FLT3-ITD+ AML cell lines (MOLM-14 and MV4-11) and three FLT3-mutant AML patient samples cultured in HS5 stromal cell conditioned media that models bone marrow microenvironment (BME) induced TKI resistance. In MOLM-14s, FF-10101 more effectively suppressed rebound ERK re-activation after 24h drug exposure than gilteritinib, potentially explaining this enhanced activity. Nevertheless, MOLM-14 and MV4-11s with NRAS G12C and Q61K co-mutations exhibited significant resistance to FF-10101 and gilteritinib. Conclusions: FF-10101 is active against nearly all mutations associated with FLT3 TKI resistance except for C695 mutations (site of FF-10101 binding) and the gilteritinib/crenolanib-resistant Y693C mutation (which has yet to be identified clinically). FF-10101 potentially overcomes resistance associated with the BME but likely remains vulnerable to RAS activating mutants. In report of phase 1 study of FF-10101 in relapsed/refractory AML (NCT03194685), 8/30 evaluable patients exhibited response (27%) with five responding patients previously receiving a FLT3 TKI, including one CRi after progression on gilteritinib. These results and our findings show that FF-10101 has potential to benefit AML patients that acquire certain FLT3 TKD resistance mutations or exhibit progression with other FLT3 TKIs, including gilteritinib. Citation Format: Timothy T. Ferng, Daisuke Terada, Makoto Ando, Theodore C. Tarver, Fihr Chaudhary, Kimberly C. Lin, Aaron C. Logan, Catherine C. Smith. The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms [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 LB516A.

The Irreversible FLT3 Inhibitor FF-10101 Is Active Against a Diversity of FLT3 Inhibitor Resistance Mechanisms.

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2).To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3).Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. [Display omitted] DisclosuresTyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Rapid and Sensitive Diagnosis of Drug-Resistant FLT3-F691L Mutation by CRISPR Detection.

Sensitive and efficient detection of drug-resistant mutations is essential in cancer precision medicine. In treating acute myeloid leukemia (AML), FLT3 gene F691L mutation shows universal resistance to all currently available FLT3 inhibitors. However, there is no particular detection method for FLT3-F691L. Commonly-used first-generation sequencing (FGS) approaches have low sensitivity, and next-generation sequencing (NGS) is time-consuming. Herein, we developed an accurate and sensitive FLT3-F691L diagnostic method by CRISPR detection. Briefly, the FLT3-691 region is amplified by recombinase polymerase amplification (RPA) and detected by L691-crRNA induced Cas12a reaction, and finally the result can be directly observed under a blue lamp or analyzed by a fluorescence reader. Confirmed by the tests on diluted plasmids and 120 AML patient samples, this method can achieve a sensitivity of 0.1% and complete the whole diagnosis process within 40 min. Potentially, this method will play an important role in point-of-care applications and guidance of AML treatment.

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Mechanisms of Acquired Resistance to Gilteritinib Therapy in Relapsed and Refractory FLT3 -Mutated Acute Myeloid Leukemia

Introduction: A major mechanism of clinical response to FLT3 tyrosine kinase inhibitors (TKIs) is the induction of terminal granulocytic differentiation of leukemic blasts (Sexauer, et al. Blood 2012). However, the clinical activity of many FLT3 TKIs is limited by novel, treatment-emergent activating mutations in FLT3 that confer in vitro resistance–particularly substitutions at D835 in the tyrosine kinase domain (TKD) activation loop and the so-called “gatekeeper” residue F691. Gilteritinib (ASP2215) is a highly potent and selective oral inhibitor of FLT3 that is active against both FLT3 -internal tandem duplication (ITD) and TKD mutations and is currently in phase 3 trials. As a single agent, gilteritinib shows significant clinical activity in relapsed/refractory (R/R) FLT3 -mutated AML (Perl, et al. Lancet Oncol 2017), but mechanisms of therapeutic resistance have yet to be systematically described. We therefore analyzed the serial morphologic and genetic data of patients treated with gilteritinib to better characterize response and identify treatment-emergent genetic alterations associated with disease progression.Methods: We studied adults with R/R FLT3 -mutated AML who were treated at FLT3 inhibitory doses (≥80mg/day) on a phase 1/2 study of gilteritinib at 2 institutions (CHRYSALIS trial, NCT02014558). Clinical and laboratory data were collected from chart review. FLT3 PCR and karyotype were sent at study entry and each treatment assessment, and next generation sequencing (NGS) results were recorded at study entry and completion. FLT3 mutant:wild-type (WT) allelic ratios were converted to mutation frequencies. Subjects were scored as a “differentiation response” if they had a >50% reduction in marrow blasts from baseline with granulocytic hyperplasia and a stable or increased FLT3 mutation frequency, while subjects were scored as a “response without differentiation” if they had a >50% reduction in marrow blasts with at least a 2-fold reduction in FLT3 mutation frequency. All other responses were considered “persistent disease.”Results: 30 subjects were studied. 4 were excluded due to a lack of evaluable data and 5 are currently being analyzed. Of the remaining 21 subjects, the median age was 66 years (range 22 - 87). 19/21 subjects (90%) had a FLT3 -ITD mutation at study enrollment, including 3 (14%) with both FLT3 -ITD and FLT3 -D835 mutations. Two subjects (10%) had a FLT3 -D835 mutation only. 10/21 subjects (48%) had a “differentiation response,” 7/21 subjects (33%) had a “response without differentiation”, and 4/21 subjects (19%) had persistent disease on gilteritinib. The median duration of gilteritinib therapy was 154 days (range 42-536+ days). One subject remains on gilteritinib at the time of data collection and 16 have progressed, with 4 withdrawn for transplant or toxicity. 9/19 subjects (47%) with serial cytogenetic data developed new cytogenetic abnormalities during therapy compared to pre-study karyotypes, including a new BCR-ABL1 fusion at progression in 1 subject. Cytogenetic evolution did not always associate with disease progression. New somatic mutations developed on gilteritinib in 7/16 progressing subjects and 1 non-progressing subject on a pre-transplant marrow (50%), including mutations in NRAS (n=4), IDH2 (n=1), CBL (n=1), and TBL1XR1 (n=1). 3 patients acquired FLT3 -F691L mutations at progression. Two patterns of resistance to gilteritinib were identified based on NGS data: 1) “clone swapping” in which the original FLT3 mutation is lost with the concurrent acquisition of mutations in other genes and 2) “clonal evolution” in which baseline mutations in FLT3 and other genes persist but additional mutations are acquired, causing subclonal expansion (Figure 1).Conclusions: Most patients responding to gilteritinib maintain a stable marrow FLT3 mutant allele burden, indicating terminal differentiation of leukemic blasts. Treatment-emergent mutational activation of signal transduction underlies the majority of cases of gilteritinib resistance, but only a small number-all F691L-are new FLT3 mutations. Cytogenetic clonal evolution during gilteritinib occurs commonly but is not always associated with resistance. Mechanisms of gilteritinib resistance include the acquisition of new driver mutations and selection for FLT3 -WT subclones. Dual targeting of NRAS signaling and FLT3 may prove useful clinically. [Display omitted] DisclosuresMorrissette:Novartis: Consultancy. Shah:Daiichi-Sankyo: Research Funding; ARIAD: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding. Smith:Astellas Pharma: Research Funding; Plexxikon Inc.: Research Funding. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding. Perl:Novartis: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Pfizer: Other: Advisory Board; Asana Biosciences: Other: Scientific advisory board; Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Astellas: Consultancy.

The Pan-FLT3/BTK Multi-Kinase Inhibitor CG '806 Induces AML Killing in FLT-Mutant and Wild Type Cells, and Exerts Synergistic Pro-Apoptotic Effects with Concomitant Targeting of Anti-Apoptotic Bcl-2 and/or Mcl-1

Internal tandem duplication (ITD) or point mutations of the Fms-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with acute myeloid leukemia (AML) (Thiede et al., 2002). The ITD mutation is associated with very poor prognosis (Kottaridis et al., 2003) and additional acquired mutations of FLT3, including the D835 or the “gatekeeper” F691 mutation, have been identified in patients who developed resistance to currently used FLT3 inhibitors (Man et al., 2012; Smith et al., 2012). In addition, the upregulation of FLT3 ligand (FL) resulting from conventional chemotherapy, or bone marrow (BM) microenvironmental-mediated protection of AML cells, are also associated with resistance to FLT3-targeted therapy (Sato et al., 2011).CG'806 is a first-in-class inhibitor that demonstrates sub-nanomolar activity against FLT3, Bruton's tyrosine kinase (BTK), aurora kinases, and certain other kinase family clusters in cell-free systems. We evaluated the anti-leukemia effect of CG'806 in human and murine leukemia cell lines harboring FLT3-ITD mutations and determined induced cell growth inhibition and apoptosis at sub-nanomolar to low nanomolar levels. Importantly, CG'806 also exerted pronounced anti-leukemia activity against cells with D835 TKD and FLT3-ITD plus F691L, Y842D or D835 dual mutations. CG'806 triggered marked apoptosis in vitro primary AML patient samples with and without FLT3-ITD mutations. Mechanistically, CG'806 demonstrated profound suppression of phospho-FLT3, -BTK and -aurora kinases, and their downstream signaling pathways (Zhang et al., AACR Hematological Malignancies, 2017). In addition, CG'806 completely abrogated protection mediated by FL or BM stromal cells in FLT3 ITD mutated MOLM14 cells, while quizartinib and sorafenib activity was markedly reduced in co-culture with mesenchymal stromal cells (MSCs). In a Baf3-ITD cell-engrafted murine leukemia model, CG'806 (delivered orally Qdx5 each week using a primitive formulation) significantly reduced leukemia burden, eliminated leukemic blasts, and extended survival in a dose-dependent manner versus the (vehicle) control.Interestingly, CG'806 did not modulate anti-apoptotic proteins Bcl-2 levels, but upregulated anti-apoptotic Mcl-1, albeit marked cleavage of caspase-3 and PARP was observed in murine Baf3-FLT3-ITD mutated cells. Hence, we tested combinations of CG'806 plus the Bcl-2 antagonist ABT199, the Mcl-1 inhibitor A1210477, or both. All combinations showed marked synergistic pro-apoptotic effects in both FLT3 wild type (WT) and mutated leukemic cells, including those cells harboring dual or “gatekeeper” mutations (e.g., CIs were 0.41 ± 0.04, 0.26 ± 0.04, 0.59 ± 0.09 and 0.90 ± 0.02 in FLT3 WT, ITD, ITD+F691L and ITD+D835Y cells, respectively, in triple combo of CG'806, ABT199 and A1210477).In summary, CG'806 was effective in both FLT3 WT and mutant AML cells, including those harboring dual ITD plus D835/F691 mutations. CG'806 also abrogated AML cell protection mediated by FL or BM stromal cells. Concomitant blockade of FLT3 and Bcl-2 or Mcl-1 synergistically induced pro-apoptotic effects in both FLT3 WT and mutant leukemic cells including dual mutant cells. CG'806 is under development for the treatment of patients with FLT3 WT and FLT3-mutated AML. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Dual Mechanism Inhibitor Control and Pharmacological Utility of Drug Resistance FLT3-ITD in Acute Myeloid Leukemia Cells

FLT3 mutations are among the most common genetic alterations in acute-myeloid leukemia (AML). They are associated with poor prognosis. Multiple FLT3 inhibitors have been in clinical evaluation at various stages. Resistance to FLT3 inhibitors due to acquired point mutations in the tyrosine-kinase domain (TKD), have limited the effectiveness of treatments. A “gatekeeper” mutation (F691L), is also resistant to most FLT3 inhibitors. New therapies are therefore needed. FLT3 inhibitors are needed to protect against FLT3-TKD mutations and FLT3 internal tandem duplicate (FLT3–ITD). We identified KX2-391, a dual FLT3/tubulin inhibitor, and examined its efficacy and mechanisms for overcoming drug-resistant FLT3ITD-TKD mutations. KX2-391 had potent growth inhibitory effects and apoptosis promoting effects on AML cell lines that harbor FLT3-ITD mutations. KX2-391 orally administered significantly prolonged the survival time of a murine model with leukemia caused by FLT3ITD-F691L. KX2-391 also inhibited growth of primary AML cells that express FLT3ITD-F691L and 2 primary cells that are FLT3ITD-D835Y. Preclinical data suggest that KX2-391 is a promising FLT3 inhibitor. The treatment of AML patients with FLT3 mutations, particularly refractory/relapsed patients suffering from F691L or other FLT3TKD mutations.

Phase 1 Trial of Vodobatinib, a Novel Oral BCR-ABL1 Tyrosine Kinase Inhibitor (TKI): Activity in CML Chronic Phase Patients Failing TKI Therapies Including Ponatinib

Phase 1 Trial of Vodobatinib, a Novel Oral BCR-ABL1 Tyrosine Kinase Inhibitor (TKI): Activity in CML Chronic Phase Patients Failing TKI Therapies Including Ponatinib

Discovery of small molecule FLT3 inhibitors that are able to overcome drug-resistant mutations

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC50s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.

Abstract 3766: Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition

Abstract Background: More than 30% of de novo AML patients were identified with FLT3 mutations. Two kinds of mutations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, account for 23% and 7% of all AML cases, respectively. FLT3 inhibitors, which have been recommended by both NCCN and ELN guidelines, are often used as single or combo agent to treat FLT3 m+ AML. However, primary and secondary acquired resistance to FLT3 inhibitors is a remarkable challenge. It has been revealed that AXL can activate FLT3, resulting in reduction of response to FLT3 inhibitors. Furthermore, TKD mutations can also lead to development of drug resistance. Therefore, FLT3/AXL dual inhibitor with potent FLT3-TKD inhibition may provide better efficacy as novel AML treatment option. Results: JRF104 is a potent FLT3/AXL dual inhibitor with a kinase inhibition IC50 value of 0.42 nM and 1.22 nM, respectively. In vitro studies suggested that JRF104 could potently inhibit the proliferation of MV4-11 and Molm-13 cells, which endogenously express FLT3-ITD mutation. JRF104 shows wide spectrum in vitro antiproliferation activity against all FLT3 mutation types, including the “gate-keeper” F691L mutation. JRF104 can effectively reduce MDA-MB-231 cell migration in vitro, accomplished by blocking tumor migration pathway regulated by AXL. In MV4-11 xenograft model, JRF104 inhibited tumor growth at the dose of 1 mg/kg, QD (TGI: 65%); significantly reduce tumor volume at 5 mg/kg, QD (TGI: 118%). In Molm-13 xenograft model, tumor volume was well controlled at 15 mg/kg, QD (TGI: 94%), and all tumor volume reduced to zero at 50 mg/kg, QD (TGI: 104%). In CT-26 syngeneic model, JRF104 enhanced anti-tumor growth effect when combined with anti-PD-1 antibody and suppressed pERK and pAKT in tumor tissues. Additionally, JRF104 demonstrated high oral bioavailability in mouse, rat, and dog. In 28 days pre-clinical toxicology studies, JRF104 was well tolerated in all tested rats. HNSTD and NOEAL were determined to be 20 mg/kg QD and 3 mg/kg QD, respectively. All results support that JRF104 with high efficacy and safety. Conclusion: JRF104 demonstrated consistently potent inhibition of FLT3 and strong antitumor efficacy against FLT3-driven tumors in preclinical studies. Based on these datas, JRF104 may overcome resistance caused by AXL and FLT3-TKD mutation, representing a novel promising clinical antitumor agent for treating AML patients with FLT3 mutations. Citation Format: yuquan wei, Xiawei Wei, Yanzhong Zhang, Wanru Qin, Xiangyu Fu, Lihong Hu, Yuanfeng Xia, Jian Zuo, Zhong Xiong, Shanshan Yin, Shaohua Qin, Ge Dong, Ying Chen, Charles Ding, Chichung Chan, Shuhui Chen. Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3766.

Mutation Analysis of ABL1 Gene and its Relation to the Achievement of Major Molecular Response in Indonesian Chronic Myeloid Leukemia Patients

Background: Imatinib mesylate is the first tyrosine kinase inhibitor approved for chronic myeloid leukemia (CML) therapy. Imatinib is an effective drug. However, previous studies have shown that about 20-30% of patients eventually would develop resistance to imatinib. Approximately 40% of imatinib resistance is associated with BCRABL kinase domain mutation. One of the most common and serious variations account for imatinib response is T315I of ABL1 gene. Objective: The study aimed to examine the association of T315I mutation with the ABL1 gene and its relation to major molecular response (MMR) achievement in CML patients. This study also examined other mutations adjacent to T315I, i.e., F311I, F317L, and different possible variations in the ABL1 gene. Methods: This was a cross-sectional study on Indonesian CML patients in chronic phase. We analyzed 120 blood samples from patients in chronic phase who have received imatinib mesylate (IM) for ≥12 months. Results: There were no T315I, F311I, and F317L mutations found in this study. However, we found another variation, which was 36 substitutions from A to G at position 163816 of ABL1 gene (according to NG_012034.1). Conclusions: We found no T315I, F311I, and F317L mutations in this study. Our findings suggest that there might be other factors that influenced the MMR achievement in our study patients. However, there were 36 substitutions from A to G at position 163.816 (according to NG_012034.1) that needed further examination to explore the significance of this mutation in clinical practice.

The landscape of BCR-ABL mutations in patients with Philadelphia chromosome-positive leukaemias in the era of second-generation tyrosine kinase inhibitors.

BCR-ABL mutations are associated with resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia chromosome-positive leukaemia. The emergence of these mutations in the era of second-generation TKIs, such as dasatinib and nilotinib, remains an evolving field. We conducted a retrospective study to quantitatively characterize the BCR-ABL transcript and mutation status during treatment with first-generation and second-generation TKI therapies. BCR-ABL mutations were detected by direct sequencing for patients with Philadelphia chromosome-positive leukaemia receiving TKI therapies. The efficacy of TKI therapy was quantitatively assessed by calculating the log reduction of BCR-ABL transcripts, which was measured using real-time quantitative polymerase chain reaction. Fisher's exact test was performed to analyse the associations of log reduction <3 and mutation status. We found 35 patients harbouring 55 mutations of 43 different types, of which 30% occurred in patients receiving imatinib, 27% in nilotinib, and 43% in dasatinib. We found a novel germline mutation, N336 N (AAC➔AAT), and two novel frameshift mutations, Asn358Thr fs*14 and Gly251Ala fs*16. T315I was the most common missense mutation, followed by V299L and F317L. Intron 8 35-bp insertion was the most frequent frameshift mutation. Both missense and multiple BCR-ABL mutations were significantly associated with worse molecular response compared with the molecular response of patients without mutation. Missense mutations, rather than frameshift, were associated with less log reduction, while the T315I, F317L, and T315A mutations were significantly correlated with poor log reduction. Collectively, amino acid substitutions at T315I, F317L, and T315A accounted for the majority of missense mutations and the loss of major molecular response. Mutation analysis is essential for patients receiving TKI therapy who exhibit an unfavourable response. The present study provided a landscape of BCR-ABL mutations in the era of second-generation TKIs.

Gilteritinib is a clinically active FLT3 inhibitor with broad activity against FLT3 kinase domain mutations

AbstractGilteritinib is the first FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitor (TKI) approved as monotherapy in acute myeloid leukemia with FLT3 internal tandem duplication and D835/I836 tyrosine kinase domain (TKD) mutations. Sequencing studies in patients have uncovered less common, noncanonical (NC) mutations in FLT3 and have implicated secondary TKD mutations in FLT3 TKI resistance. We report that gilteritinib is active against FLT3 NC and TKI resistance-causing mutations in vitro. A mutagenesis screen identified FLT3 F691L, Y693C/N, and G697S as mutations that confer moderate resistance to gilteritinib in vitro. Analysis of patients treated with gilteritinib revealed that 2/9 patients with preexisting NC FLT3 mutations responded and that secondary TKD mutations are acquired in a minority (5/31) of patients treated with gilteritinib. Four of 5 patients developed F691L mutations (all treated at <200 mg). These studies suggest that gilteritinib has broad activity against FLT3 mutations and limited vulnerability to resistance-causing FLT3 TKD mutations, particularly when used at higher doses.

Interim Results of the Real-Life Study Evaluating the Efficacy and Safety of Ponatinib “Topase” Reveals Induction of Early Molecular Responses in Patients with TKI-Resistant or Intolerant CML

Interim Results of the Real-Life Study Evaluating the Efficacy and Safety of Ponatinib “Topase” Reveals Induction of Early Molecular Responses in Patients with TKI-Resistant or Intolerant CML