FLT3 D835Y

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.

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.

Compound 15 also possessed potent antiproliferative activities against BaF3 cells carrying various FLT3-TKD and FLT3-ITD-TKD mutations, indicating its potential to overcome on-target resistance caused by FLT3 mutations. In summary, compound 15 showed promising potential for further exploration as a treatment of AML.

In primary patient cell lines from FLT3 wildtype (WT) patients, KME-0584 inhibits leukemia stem cell progenitor function as measured by the colony formation assay in methylcellulose with higher potency than IRAK4 inhibitor compounds that lack IRAK1 activity such as CA-4948 (Emavusertib)...Given that monocytic-like subtypes of AML are resistant to Venetoclax plus Azacitidine (VEN/AZA) (S Pei et...KME-0584 exhibits superior potency and efficacy to gilteritinib in the FLT3-ITD (D835Y) xenograft model after QD oral dosing, with sufficient PK and oral bioavailability across multiple species to support QD or BID dosing in the clinic. KME-0584 does not inhibit any of the major or minor cytochrome P450 enzymes at anticipated clinical concentrations and early indication from ongoing GLP toxicology studies suggest that it could be safely administered in humans. A clinical study of KME-0584 in relapsed/refractory AML and HR-MDS is currently planned to start in 1H 2024.

The "clonal attack" by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in AML clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the "clonal attack" may be broadly applicable to the quest for cancer cure.

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.

Taken together, we present novel neoantigens with promise as immunotherapy targets for AML and other hematologic malignancies with FLT3-D835 mutations. Future studies will evaluate the efficacy of Neo-D835H-specific TCR-engineered T-cell-based immunotherapy in vivo in patient-derived xenograft murine model and TCR-based therapeutic approaches targeting other FLT3-TKD mutations.

TCR cells rejected both CD34 and CD34 AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34 AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.

In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.

Finally, the docking and simulation studies revealed the important interactions of compound 5f with wild type and mutant FLT3. The results of this study showed that some novel spiroindoline quinazolinedione compounds could be potential candidates for further development as novel targeted anticancer agents.

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.

A novel class of aminopyrimidine-based Bruton's tyrosine kinase (BTK) and FMS-like tyrosine kinase 3 (FLT3) dual-target inhibitors based on the BTK inhibitor spebrutinib was designed for the treatment of acute myeloid leukemia...Finally, intraperitoneal administration of 14m at 20 mg/kg significantly repressed the growth of MV-4-11 cells with a TGI value of 95.68% with no apparent toxicity. These BTK/FLT3 dual-target inhibitors represent promising leads for further structural optimization and antitumor mechanism studies.

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.

We compared the abilities of five clinically used FLT3-ITD inhibitors, namely, midostaurin, crenolanib, gilteritinib, quizartinib, and sorafenib, to induce apoptosis...Homoharringtonine decreases the protein levels of Mcl-1, FLT3-ITD, and Axl. Moreover, it synergistically induces apoptosis with gilteritinib in vitro and prolongs survival of MOLM-13/sor xenografts. The GSK3β/Mcl-1 axis works as the hub of FLT3-ITD inhibitors and plays a critical role in resistance against FLT3-ITD AML-targeted therapy.

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.

Based on the docking and free energy ranking, the top compound was selected for synthesis and screening. Of these three compounds, PCW-A1001 proved to be highly selective for the FLT-3 (D835Y) mutant, with an IC of 764 nM, whereas the IC of FLT-3 WT was 2.54 μM.

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.

Seventy percent of enrolled patients had more than 3 prior anti-leukemic treatment attempts, and four patients had relapsed or refractory disease after treatment with other FLT3 inhibitors including gilteritinib, quizartinib, or HM43239. A dose-escalating phase 1a clinical data of PHI-101, which reflects up to cohort 4 of the study, indicates that PHI-101 generated potent FLT3 inhibition leading to encouraging anti-leukemic responses in R/R AML patients, including in those with prior FLT3 TKI therapeutic failure. PHI-101 showed good tolerance and favorable safety profile and reduced leukemic blasts significantly with 28-day dosing. PHI-101 sustained its activity to clear FLT3-ITD and/or FLT3-TKD mutations including D835Y or N676K identified in AML patients.

However, hematologic remissions occur in only 10-30% (HMA monotherapy) or 40-70% (HMA + Venetoclax) of pts, and initially responsive disease will eventually develop secondary resistance. In an attempt to decipher non-genetically mediated resistance mechanisms, we studied DNA methylation profiles and observed relative hypomethylation at resistance. Further studies focusing on epigenetic alterations towards secondary HMA resistance are warranted.

Autophagy might be stimulated by acquired mutation or BME, and bypass activate FLT3 downstream signaling to mediate FLT3 inhibitor resistance in FLT3-ITD-positive AML. Targeting autophagy could be a promising strategy to overcome resistance.

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.

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).

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.

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...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.

Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP, that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC = 43.8, 97.2, and 92.5 nM respectively)...4ACP did not show cytotoxic effects on normal BNL and H9c2 cells and demonstrated decreased activity against c-Kit enzyme, hence, indicating lower probability of synthetic lethal toxicity and a relatively safer profile. In light of these data, 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML.

Tyrosine kinase inhibitors, such as quizartinib (AC220) and midostaurin (PKC412), markedly decrease FLT3-ITD retention and increase PM levels of the mutant. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

AML with RUNX1-RUNX1T1 fusion gene is currently not indicated for transplantation in the first remission. Previous studies have demonstrated that approximately 30% of patients with CBF-AML harbored the KIT mutations at diagnosis, which might be an indicator of poor prognosis. In our study, the KIT mutations were detected much more frequently than in previously studies of newly-diagnosed CBF-AML.

Upon engraftment, treatment was started with either chemotherapy (cytarabine for 5 days + doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Taken together, these results show that the specific treatment given not only affected survival of the FLT3 N676K mutated KMT2A-MLLT3 leukemia, but also impacted how the genetically distinct cells evolved. The general lack of acquired mutations upon targeted treatment suggests that target-independent mechanisms that result in alternate activation of survival/proliferation explains acquired resistance in a majority of mice and provides novel insights into treatment resistance.

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.

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.

CD56 expression was the most common aberrancy seen in more than 25% cases. CD34negativeHLADR negative is highly associated with NPM1 mutation.

Our results demonstrate that CCT245718 is a potent dual FLT3/Aurora A inhibitor that can overcome TKD-mediated acquired resistance.

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.

Subsequently the patient underwent three consolidation cycles with HD Ara-C and midostaurin (days 8-21). It is important to note that the patient had remained free from leukaemia during three decades and a half, with AML developing only after acquisition of two other gene mutations (NPM1 and FLT3 TKD). These observations shed light on the paradigm of multiple genetic hits hypothesis of leukemogenesis and, after demonstration of the same IDH1 (R132H) mutation in chondroma tissue, prompt us to monitor the patient rather than candidate him to alloHCT in I CR.

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.

Upon engraftment, treatment was started with either chemotherapy (cytarabine for 5 days (d)+doxorubicin for 3 d), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Conclusion The specific treatment given affected survival and impacted the evolution of genetically distinct cells. The general lack of acquired mutations upon targeted treatment suggests that target-independent mechanisms that result in alternate activation of survival/proliferation explains acquired resistance in a majority of mice.