FLT3 F691L

Recently, several FLT3 inhibitors have demonstrated clinical activity and three are currently approved - midostaurin, quizartinib, and gilteritinib. In summary, compound 24 has inhibition potency on FLT3 comparable to gilteritinib, but a more balanced inhibition on FLT3 secondary mutations, especially FLT3-ITD/F691L which is gilteritinib resistant. Compound 24 may serve as a promising lead for the drug development of either primary or relapsed AML with FLT3 secondary mutations.

Introduction: Luxeptinib (CG-806; LUX) is an orally active noncovalent kinase inhibitor of Bruton's tyrosine kinase (BTK), wild type and mutant forms of Fms-like tyrosine kinase 3 (FLT3) (including the internal tandem duplicates (ITD), tyrosine kinase domain (TKD), and F691L mutant forms), and growth receptors like KIT, CSF1R, PDGFRα, and TRKs. LUX has a favorable safety profile in patients at all tested dose levels, for multiple cycles, for both studies. Antitumor activity was observed in a heavily pretreated relapsed AML patient and in multiple B-NHL subtypes and CLL/SLL patients including several with prior ibrutinib exposure. Continuous dosing of patients with R/R AML and Higher-Risk MDS with the G3 formulation is ongoing; with updated clinical data (200 mg dose level) to be presented at the meeting.

Our results show that GNF-7 is a potent FLT3-ITD inhibitor and may become a promising lead compound applied for treating some of the clinically drug resistant patients.

More importantly, 22f showed single-digit nanomolar GI values in the mutant FLT kinase expressed Ba/F3 cell lines including FLT-D835Y (GI = 0.29 nM) and FLT3-F691L (GI = 2.87 nM). Molecular docking studies indicated that the compound exhibits a well-fitted binding mode as a type 1 inhibitor in the homology model of active conformation of FLT3 kinase.

Studies indicate that it mediates proapoptotic effects on cells by inhibiting FLT3 and BCR-ABL pathways, and other possible targets. Compound 1 is more potent against FLT3-ITD than BCR-ABL, and it may have other possible targets; however, compound 1 is first step for further optimization for the development of a balanced FLT3-ITD/BCR-ABL dual inhibitor for the treatment of relapsed FLT3-ITD mutated AML with multiple secondary clinical resistant subtypes such as FLT3-ITD/D835Y, FLT3-ITD/F691L, and cells co-expressing FLT3-ITD and BCR-ABL.

While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition...Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells... This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

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). 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.

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.

Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC: 0.62 nM), starting from FN-1501...Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T = 3.5 h in rat and T = 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.

We tested CTS2016 as a single agent or in combination with either venetoclax, a Bcl-2 inhibitor, or azacitidine, a hypomethylating agent (HMA) and an epigenetic modulation drug, in a series of in vitro and in vivo studies using various AML models. CTS2016 resulted in a potent growth inhibitory effect with strong induction of cell death in a spectrum of AML cell lines carrying FLT3 mutations (FLT3-ITD and/or FLT3-TKD). CTS2016 orally administered once daily, demonstrated potent and dose-dependent antitumor responses in a variety of AML xenograft mouse models. These data demonstrate that CTS2016 could be a promising therapy for treating patients diagnosed with AML and MDS harboring FLT3 mutations and solid tumors such as NSCLC and TNBC with high-expression of AXL. More proof-of-concept data would come from the ongoing clinical trial.

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. 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.

Gilteritinib is a highly selective and effective inhibitor of the FLT3/ITD mutated protein, and is used successfully in treating acute myeloid leukaemia (AML)...Both mutants show a lower activation energy barrier which suggests that they are more likely to adopt an active state until inhibited, making the mutant enzymes more active. This suggests that a higher efficiency of tyrosine kinases contributes to resistance not only against type 2 but also against type 1 kinase inhibitors.

Our study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

Moreover, the treatment with compound 13 led to robust inhibition of FLT3 autophosphorylation on Tyr589/591 in MV4-11 cells. In summary, our data demonstrated that 13 was worthy of further study for the treatment of AML.

Luxeptinib suppresses additional signaling pathways in AML cells (CSF1R, PDGFRα, TRK, SYK, BTK, LYN, AKT, ERK, MAPK), kills primary AML cells insensitive to other FLT3 inhibitors at pM and low nM concentrations, and shows enhanced activity in combination with venetoclax...At 450 mg BID, one heavily pretreated AML patient (received 8 prior regimens including alloSCT and FLT3 inhibitors gilteritinib and crenolanib) had a 99% reduction in peripheral blasts (6.4x103/µL at C1D1 to 0.1x103/µL at C1D15), though the blast proportion increased during Cycle 2... As of July 8, 2022, luxeptinib is well tolerated at dose levels of 450, 600, 750, and 900 mg BID over multiple cycles without evidence of having reached the MTD. Luxeptinib in heavily pretreated relapsed FLT3-ITD AML patients has shown anti-tumor activity including a durable, MRD negative CR. Enrollment of patients with R/R AML is ongoing and updated clinical data with the G3 formulation will be presented at the meeting.

Several FLT3 inhibitors have been developed such as the first generation multikinase inhibitor Midostaurin which is approved for newly diagnosed FLT3 mutant AML in combination with induction chemotherapy...Herein we describe 2082-0047, a novel tyrosine kinase inhibitor with sub-nanomolar potency against FLT3 mutant AML, including TKD mutations...Our non-GLP in vivo safety studies confirmed a broad therapeutic window of 2082-0047. Ongoing Investigational New Drug (IND) enabling studies will include comparing the effect of 2082-0047 to gilteritinib in an immunocompetent syngeneic AML murine model harboring both a Flt3-ITD and F691 TKD gatekeeper mutation.

Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.

Pluripotin treatment curbed the progression of AML in multiple in vivo models including patient-derived primary AML cells in mouse xenotransplants. As a proof of concept, we demonstrate that targeted polypharmacological inhibition of key signaling nodes driving adaptive resistance can provide a durable response.

Moreover, the per-residue free energy decomposition suggested that the four residues (Phe803, Gly831, Leu832, and Ala833) located at the A-loop of FLT3 had a significant impact on the binding affinity of gilteritinib to the F691L mutant. This study may provide useful information for the design of novel FLT3 inhibitors specially targeting the F691L gatekeeper mutant.

The combination of crenolanib and sorafenib was tolerable with antileukemic activities and rare emergence of FLT3-TKD mutations, which warrants further investigation.

The selectivity profiles of compound 13a in the oncology kinase panel and various human cancer cell lines were prominent, demonstrating that its inhibitory activities were mainly focused on a few members of the receptor tyrosine kinase family and AML versus solid tumor cell lines. Furthermore, significant in vivo anticancer efficacy of compound 13a was confirmed in a xenograft animal model implanted with FLT3-ITD/D835Y-expressing MOLM-14 cells related to secondary TKD mutation.

Of 23 pts with R/R AML (median 3 prior Rx, 78% with ≥1 prior FLT3i including prior gilteritinib in 70%, and 39% had a prior alloSCT), 78% achieved CRc (3 CR, 15 CRi) with 6/16 and 5/18 responders achieving FLT3-PCR and multicolor flow cytometry negativity, respectively. Interestingly, RAS/MAPK mutations but not emergent TKD mutations were associated with primary and secondary resistance to the triplet. Accrual continues and updated clinical, NGS and mass cytometry (CyTOF) data will be presented.

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. 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).

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.

Acquisition of multiple Ras/MAPK pathway gene mutations at relapse suggests a high level of pathway reactivation is needed to overcome the gilteritinib treatment effect. These findings provide insight into the R/R AML molecular profile and the impact of FLT3 inhibitors on mutational evolution associated with treatment resistance and benefit of gilteritinib across a wide spectrum of molecular and genetic subgroups in FLT3-mutated R/R AML.

No clear myelosuppression was observed in the treated group of 36 in the MPO strain of zebrafish, even at 10 μM. In summary, our data demonstrated that 36 may represent a promising candidate for the treatment of FLT3 mutant AML.

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.

We have recently shown for the first time that a novel MCL-1 inhibitor S63845 elicited synergistic activity with FLT3 inhibitors AC220, sorafenib and with a multi-kinase inhibitor midostaurin at nanomolar doses in pre-clinical in vitro models of FLT3-ITD AML, including cells resistant to venetoclax ( Skwarska A, et al...Mice were treated for 3 weeks with low doses of midostaurin (25 mg/kg/5 days per week) and with MIK665, structurally optimized version of S63845 Mcl-1 inhibitor with improved pharmacokinetics in mice ( Halilovic E, et al...The long-term effect of combination on mice survival is currently being tested and will be reported. Altogether, our results indicate that efficacy of FLT3-ITD inhibitors can be enhanced through combination with low doses BH3 mimetics targeting MCL-1 and provide a rationale for the clinical evaluation of such combinations in FLT3 mutant AML patients.

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. 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.

This is the first report to analyze R/R and ND unfit AML cases in a Japanese cohort using F1H NGS, revealing a higher incidence of FLT3 -ITD/TKD mutations than previously reported. Therefore, F1H mutational analyses for R/R and ND unfit AML patients harboring FLT3 -ITD/TKD mutations may reveal novel therapeutic targets that are sensitive to FLT3i. Samples from these patients showed non-canonical gain-of-function mutations, such as N676K, S451F, V592D, and F691L, which could guide the selection of optimal anti-FLT3 therapies.

Introduction Despite the advent of targeted therapy for FLT3 -mutated AML, unmet need still exists for patients unfit for intensive chemotherapy, with no evidence that overall survival (OS) can be improved by combining either venetoclax (Konopleva et al...Adaptive on-target gilteritinib resistance may be due to the FLT3 -F691L gatekeeper mutation, whereas off-target resistance may be due to loss-of-function variants in CBL , which encodes an E3 ubiquitin-protein ligase that negatively regulates FLT3 (McMahon et al , 2019)...Preliminary efficacy was observed in CBL -mutated patients. Further clinical investigation of this regimen is warranted in patients with FLT3- or CBL -mutant AML.

Luxeptinib simultaneously suppresses additional signaling pathways in AML cells (CSF1R, PDGFRα, TRK, SYK, BTK, LYN, AKT, ERK, MAPK), kills primary AML cells insensitive to other FLT3 inhibitors at pM and low nM concentrations, and shows enhanced activity in combination with venetoclax...One heavily pretreated AML patient (8 prior regimens including alloSCT and FLT3 inhibitors gilteritinib and crenolanib) had 99% reduction of blasts in peripheral blood (from 6.38x10 3 /µL at C1D1 to 0.09x10 3 /µL at C1D15), though the decrease in blasts reversed during Cycle 2...MRD-negative complete remission (CR) was confirmed in one FLT3-ITD AML patient (6 prior regimens including 2 alloSCT and FLT3 inhibitor sorafenib) at Cycle 5 of treatment evidenced by reduction of FLT3-ITD VAF and blasts in bone marrow to below limit of detection (LOD) of PCR-based FLT3-ITD assay and high-sensitivity flow cytometry (LOD < 0.1%), respectively... As of June 7, 2021, luxeptinib is well tolerated at dose levels of 450 and 600 mg BID over multiple cycles and escalated to 750 mg BID (Cohort 3). Pharmacodynamic studies documented inhibition of FLT3 signaling, and anti-leukemic activity has been observed in heavily pretreated relapsed FLT3-ITD AML patients as evidenced by significant reduction of FLT3-ITD VAF and blasts in bone marrow and / or peripheral blood. One FLT3-ITD AML patient has had confirmed MRD negative CR and continues treatment.

Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model...Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

KX2-391 exhibited potent growth inhibitory and apoptosis promoting effects on diverse AML cell lines harboring FLT3-ITD mutations and AC220-resistant mutations at the D835 and F691 residues in TKD and inhibited FLT3 phosphorylation and its downstream signaling targets. KX2-391 also significantly inhibited the growth of 4 primary AML cells expressing FLT3-ITD and 2 primary AML cells expressing FLT3-ITD-D835Y. Our preclinical data highlight KX2-391 as a promising FLT3 inhibitor for the treatment of AML patients harboring FLT3 mutations, especially refractory/relapsed patients with F691L and other FLT3-TKD mutations.

Luxeptinib simultaneously suppresses additional signaling pathways in AML cells (CSF1R, PDGFRα, TRK, SYK, BTK, LYN, AKT, ERK, MAPK), kills primary AML cells insensitive to other FLT3 inhibitors at pM and low nM concentrations, and shows enhanced activity in combination with venetoclax...One heavily pretreated patient with relapsed AML with mutated NPM1, DNMT3A, and FLT3-ITD who had previously progressed after chemotherapy, alloSCT, and two FLT3 inhibitors (gilteritinib, crenolanib) demonstrated clinical anti-leukemic activity with a decrease in peripheral blood blasts from 93% to 10% during Cycle 1...Pharmacodynamic studies documented inhibition of FLT3 signaling pathway, and anti-leukemic activity has been observed in the setting of AML relapsed following therapy with multiple prior FLT3 inhibitors. Enrollment of patients with R/R AML continues and updated clinical data from this dose escalation study will be presented at the meeting.

AML patients are not likely to benefit from a quizartinib/pexidartinib rotation protocol. A combination of tyrosine kinase inhibitors (with different molecular targets) might be more useful in the future. Development of specific FLT3 inhibitors that are less sensitive to resistance mutations might also lead to a better outcome.

As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients.Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML.

Finally, we have found that low doses (5-50nM) of the potent PARPi, Talazoparib (Tal), in combination with low dose (10nM) of the TKIs, Quizartinib (Quiz) or Gilteritinib (Gilt), is synergistically lethal in the otherwise TKI-resistant cells. This suggests a novel model whereby Tal may resensitize cells to TKI by decreasing PARP1 effects on pSTAT5 activity which feeds back to reduce FLT3 receptor activation. Therefore, this combination has potential for treatment of TKI-resistant AML and studies are underway to translate these findings with the intent of developing an effective therapy for patients with this disease.

We also show the efficacy of LT‑171-861 in a subcutaneous implantation model and a bone marrow engrafted model in vivo, where administration of LT-171-861 led to almost complete tumor regression and increased survival. Overall, this study not only identifies LT-171-861 as a potent FLT3 inhibitor, but also provides a rationale for the upcoming clinical trial of LT-171-861 in patients with AML and FLT3-ITD mutations.