FLT3-mutated Acute Myeloid Leukemia Research Articles

Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway. Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism. Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo. Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log2 (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC50 of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC50 of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use. Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

V-FAST Master Trial: Subgroup Analysis of Outcomes with CPX-351 Plus Midostaurin in Adults with Newly Diagnosed Acute Myeloid Leukemia By FLT3 Mutation Type

CPX-351 (United States: Vyxeos®; European Union/United Kingdom: Vyxeos® liposomal), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in patients aged ≥1 year in the United States and in adults in the European Union/United Kingdom. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7+3 chemotherapy, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin (MID). V-FAST (Vyxeos - First Phase Assessment with Targeted Agents) is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial (NCT04075747) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (MID, venetoclax, enasidenib). Eligible adults in the CPX-351 + MID cohort were aged 18 to 75 years, had newly diagnosed AML (de novo AML or secondary AML) with a FLT3 mutation, were fit for intensive chemotherapy, and had an ECOG performance status of 0 to 2. The dose-exploration phase (3+3 design) determined a recommended phase 2 dose of CPX-351 100 units/m2 (daunorubicin 44 mg/m2 + cytarabine 100 mg/m2) on Days 1, 3, and 5 + MID 50 mg twice daily on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose. Herein, we report outcomes for a subgroup analysis based on the presence of a FLT3 internal tandem duplication (ITD) and/or tyrosine kinase domain (TKD) mutation within the cohort of adults primarily with de novo AML who were treated with CPX-351 + MID in the V-FAST trial. A total of 23 patients had sufficient data available to be included in the general safety and efficacy analyses in this ongoing trial at the time of the data cutoff on March 22, 2022. Of the patients included in this analysis, 18 patients had a FLT3 ITD mutation and 6 patients had a FLT3 TKD mutation; 1 patient had both FLT3 mutation types and was included in both subgroups. Patient baseline characteristics are shown in the Table. A diagnosis of de novo AML was reported for 78% of patients with FLT3 ITD mutation and all (100%) patients with a FLT3 TKD mutation. Treatment-emergent adverse events (TEAE) occurring in >50% of patients with a FLT3 ITD or TKD mutation, respectively, included febrile neutropenia (78%; 83%), nausea (67%; 67%), alanine aminotransferase increased (61%; 33%), leukopenia (61%; 50%), and thrombocytopenia (56%; 67%). All patients experienced a ≥3 grade TEAE, primarily hematologic events and infections. Serious TEAEs were reported for 6 (33%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation; no serious TEAE was reported more than once. Four (22%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation died during the study; there were no deaths on or before study Day 60. Complete remission (CR) was achieved by 14/17 (82%) patients with a FLT3 ITD mutation and by 5/6 (83%) patients with a FLT3 TKD mutation (Figure). Among patients in CR and with a measurable residual disease (MRD) assessment by multicolor flow cytometry, MRD negativity was achieved by 5/10 (50%) patients with a FLT3 ITD mutation and 1/3 (33%) patients with a FLT3 TKD mutation, all after the first induction cycle. Median (interquartile range) recovery times to an absolute neutrophil count ≥500/μL and platelet count ≥50,000/μL, respectively, were 35 (34, 43) and 34 (28, 36) days for patients in CR with a FLT3 ITD mutation and 30 (29, 35) and 30 (29, 40) days for those in CR with a FLT3 TKD mutation. A total of 10/18 (56%) patients with a FLT3 ITD mutation and 2/6 (33%) patients with a FLT3 TKD mutation have proceeded to hematopoietic cell transplantation to date. In conclusion, preliminary results from the V-FAST trial suggest the combination of CPX-351 + MID is feasible with a manageable safety profile and promising remission rates in adults with newly diagnosed, FLT3-mutated AML. Although conclusions are limited by the small numbers of patients in each subgroup, results are generally consistent for patients with FLT3 ITD and TKD mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gilteritinib Activity in Refractory or Relapsed FLT3-Mutated Acute Myeloid Leukemia Patients Previously Treated By Intensive Chemotherapy and Midostaurin: A Study from the French AML Intergroup ALFA/Filo

Background The recent phase 3 ADMIRAL trial has shown that outcomes of relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) patients were improved by the single-agent FLT3 tyrosine kinase inhibitor gilteritinib as compared with salvage chemotherapy. In this trial, overall survival (OS) was significantly improved in the gilteritinib arm and complete remission (CR/CRi), after excluding those obtained after allogenic hematopoietic stem cell transplantation (Allo-HSCT) were 26.3% in the gilteritinib group. Methods Here, the real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients (Cohort A) from the French early access program ELEGANCE between March 1st, 2019, and March 1st, 2021. Gilteritinib was administered to 27 patients in association with other treatments, while 140 received it as single agent (Cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (Cohort C). Results Characteristics of the three cohorts at gilteritinib initiation are described in Table 1. Briefly, in the whole cohort A, there were 141 (84.4%) cases of de novo AML, FLT3 mutation was ITD in 104 (62.3%) and TKD in 33 (19.8%), whereas 36 (21.6%) patients were FLT3 wild-type at diagnosis, acquired mutation later in disease evolution and were FLT3 mutated at gilteritinib initiation. Finally, 6 (3.6%) patients had both FLT3-ITD and TKD mutations. The median FLT3-ITD/wt ratio was 0.50 (IQR, 0.12-0.70 and range 0.01-1.90), and the median size of ITD was 48 bp (IQR, 30-75, range 3-408). Most patients (136, 81.4%) had an intermediate cytogenetic risk, and 84 (50.3%) had an NPM1 co-mutation. The main differences in patient characteristics in this study, compared to ADMIRAL, were ECOG ≥ 2 (83.6% vs 16.6%), FLT3-TKD mutations (21.0% vs 8.5%), primary induction failure (15.0% vs 40.0%) and previous lines of treatment (beyond 2nd in 37.1% vs 0.0%). The rates of response to gilteritinib are described in Table 1. Moreover, CRc (CR + CR without hematological recovery + CR with incomplete platelet recovery) to gilteritinib as single agent in subgroups defined as per AML phase at gilteritinib initiation were 4.5% (N=22) for patients refractory after front-line, 37.5% (N=32) for those relapsing less than 6 months from CR/CRi, 26.7% (N=30) when relapse occurred after 6 months and 16.1% (N=56) for patients refractory after 1st relapse and beyond. Finally, 32 (19.2%), 25 (17.9%) and 15 (22.4%) patients received an Allo-HSCT after gilteritinib, in CRc for 21 (65.6%), 17 (68.0%) and 11 (73.3%) patients in cohorts A, B and C, respectively. In terms of safety, in the subgroup of patients treated with gilteritinib as single agent (Cohort B), 39 (28.3%) experienced a serious adverse event (SAE) during gilteritinib treatment: infection in 22 (56.4%), hemorrhage in 1 (2.6%), cardiovascular in 4 (10.3%), hepatic in 4 (10.3%), differentiation syndrome in 2 (5.2%) and other SAE in 6 (15.4%). In terms of health care resource consumption, the median duration of hospitalization during the first 6 months of treatment was 9.0 days (IQR 0-29, range 0-117) with a median of 7 RBC units (IQR 0-14, range 0-54) and of 5 platelet units (IQR 0-13.5, range 0-56) transfusions. After a median follow-up of 14.5 months, median OS and 12-month OS were 6.4 months (IQR, 3.2-14.7) and 31.4% (95%CI 22.4-39.7) in the 140 patients who received gilteritinib as single agent, and 7.8 months (IQR, 3.2-20.8) and 38.3% (95%CI 26.0-50.4) in the subgroup of 67 patients previously treated by intensive chemotherapy and midostaurin (Figure 1). Multivariate analyses disclosed female gender (HR 1.61, 95%CI 1.07-2.42, p=0.02), adverse cytogenetic risk (HR 2.52, 95%CI 1.24-5.13, p=0.01) and allo-HSCT after gilteritinib (HR 0.13, 95%CI 0.05-0.37, p<0.0001) as factors significantly and independently associated with OS. Conclusion Although patients in this study were more heavily pretreated, these real-world data reproduce ADMIRAL results and provide new insights in the outcome of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omic Single-Cell Sequencing Reveals Genetic and Immunophenotypic Clonal Selection in Patients With FLT3-Mutated AML Treated With Gilteritinib/Venetoclax

Background: The combination of the FLT3 inhibitor gilteritinib with the BCL-2 inhibitor venetoclax (VenGilt) is highly efficacious in relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML). In a recent phase 1b trial, VenGilt demonstrated a modified complete response rate of 75% (Daver et al, J Clin Oncol. 2022; published online). However, many patients (pts) relapsed after VenGilt. Proposed resistance mechanisms include RAS pathway mutations (Ven and Gilt) and monocytic immunophenotype (Ven). Using single cell (SC) multi-omic profiling, we sought to identify drivers of resistance to this combination. Methods: We analyzed 35 serial samples from 12 pts with R/R AML treated with VenGilt on the phase 1b trial (NCT03625505). We performed high-throughput SC DNA sequencing (Mission Bio Tapestri) on all samples and concurrent multi-omic SC cell-surface immunophenotyping on 18 samples. Results: We analyzed 107,528 cells (median 8960 cells/pt) from 12 pts with 2-5 serial timepoints. Eight pts had previously received tyrosine kinase inhibitors (TKI). Nine pts had clinical responses to VenGilt with a median relapse-free time of 8.3 mo. Of these 9 pts, 3 proceeded to transplant. Each pt had ≥1 genetically distinct clone identified (median 4.5, range 2-6). Fourteen FLT3mut clones were detected, with 2 pts having 2 separate FLT3mut clones. In all 12 pts, the FLT3mut clones were reduced with VenGilt. In 7 pts with a total of 9 clones, the FLT3mut population decreased to an undetectable level. Three pts had FLT3-IDH1/2 co-mutated clones, and the double mutant clones were reduced to undetectable levels in all 3. One pt developed a dominant IDH2mut clone at relapse without a FLT3 co-mutation. Nine pts demonstrated an initial decrease in non-FLT3mut clones, including clones with DNMT3A, NPM1, IDH1/2, TET2, EZH2, WT1, and NRAS mutations, suggesting some activity of VenGilt against non-FLT3mut populations. In 3 pts, all clones aside from those associated with clonal hematopoiesis of indeterminant potential (CHIP) were undetectable at follow-up. Five pts had ≥1 pre-existing NRAS- or KRAS-mutated clone. In 4/5 pts, the RASmutclone expanded on therapy and was dominant at relapse in 3/4 pts, with the fourth pt having a dominant IDH2mut clone. All 4 pts with expanding RASmutclones had prior TKI, while the pt whose RASmutclone decreased did not. Pre-existing RAS populations were small (0.001-18.1% of total cells) and were undetectable by bulk sequencing in 2/4 cases. RAS was not co-mutated with FLT3 in the same single cell in any cases. In 2 pts, FLT3mut clones present before VenGilt initially decreased but subsequently expanded on therapy. No de novo mutations within resistant/expanding FLT3mut clones were detected. We demonstrated immunophenotypic evolution for individual genotypes, including both FLT3mut and FLT3WT clones. For 5 pts with serial multi-omic SC samples, increased CD11b and CD13 expression was observed over time on a single cell basis in ≥1 genotype within individuals, suggesting increased monocytic-like features. These features were observed among 4 RASmut, 2 FLT3mut, and 1 TET2mut clones. At baseline, these 5 pts had heterogenous immunophenotypic profiles, with genetic clones occupying monocytic, immature, and mixed phenotypes; however, all 5 pts demonstrated ≥1 clone with increased monocytic immunophenotypic features after VenGilt treatment. Conclusion: Multi-omic SC sequencing enabled direct measurement of genetic and immunophenotypic clonal architecture and evolution with VenGilt treatment. VenGilt was highly effective against FLT3-mutated clones, even in the presence of co-mutations. Genetic resistance was associated with outgrowth of pre-existing subclones, primarily RAS mutations, rather than acquistion of de novo mutations. This differs from prior studies of Gilt monotherapy, in which both outgrowth of parallel clones and secondary mutations within the FLT3-mutated clone were associated with resistance. These data suggest the addition of Ven may suppress secondary resistance mutations, possibly due to enhanced apoptosis and cytotoxicity against FLT3-mutated cells. Multiple clones, predominatly those with a RAS mutation, developed monocytic features under VenGilt pressure. It is unknown whether monocytic immunophenotype is an independent resistance mechanism or a bystander to other mechanisms, such as increased MCL1 or RAS pathway gene expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

19S Proteasome Subunits As Oncogenes and Prognostic Biomarkers in FLT3-Mutated Acute Myeloid Leukemia (AML)

19S Proteasome Subunits As Oncogenes and Prognostic Biomarkers in FLT3-Mutated Acute Myeloid Leukemia (AML)

Venetoclax Plus Hypomethylating Agent (HMA) Based Induction for Treatment-Naive Older/Unfit Patients with FLT3-Mutated Acute Myeloid Leukemia (AML)

Introduction: FMS-like tyrosine kinase 3 (FLT3) mutation (m) is one of the most prevalent mutations in acute myeloid leukemia (AML), and it is associated with an increased risk of relapse and decreased overall survival (OS). The standard of care therapy for physically fit treatment naïve patients is intensive chemotherapy with FLT3 inhibitor (i) (midostaurin). For older/unfit patients, venetoclax-based induction is preferred as per NCCN guidelines. We explored the clinical outcome of FLT3m AML patients who were ineligible for intensive chemotherapy and treated with a hypomethylating agent (HMA) plus venetoclax (Ven) based induction at Mayo Clinic. Methods: Thirty FLT3m AML patients who were ineligible for intensive chemotherapy and received HMA+Ven induction at Mayo Clinic between June 2019 and May 2022 were analyzed for clinical characteristics and treatment outcomes. Patients who received HMA+Ven for the relapsed or refractory disease were excluded. All patients received at least one cycle of HMA, either azacitidine (75 mg/m2 from day 1 to 7) or decitabine (20 mg/m2 from day 1 to 5). Responses were assessed according to the 2022 European Leukemia Net (ELN) criteria. Results: The baseline characteristics are summarized in Table 1. Most of the patients (n=27, 90%) were ≥ 65 years old with a median age of 74 years (range, 59 to 88). Majority of the patients (n=16, 53%) had de novo AML, 8 (27%) patients had secondary AML, 4 patients (13%) had primary AML with MDS-related changes, and 2 patients (7%) were diagnosed with therapy-related AML. Two-thirds of the patients (n=20, 67%) had diploid cytogenetics (CG) and 10 (33%) patients had additional cytogenetic (CG) abnormality at diagnosis. Twenty-six (87%) and 4 (13%) patients had FLT3 ITD and TKD mutation subtypes, respectively. The median baseline FLT3m allelic ratio was 0.39 (range, 0.1-0.7). Most frequent reported mutations (≥ 5%) were DNMT3A (40%), NPM1 (37%), spliceosome mutations (SF3B1, U2AF1, and SRSF2; 30%), RUNX1 (23%), and TET2 (17%). Seventy percent of the patients achieved complete remission with or without count recovery (CR/CRi) (n=21), 13% (n=4) of the patients had partial remission, and 17% (n=5) of the patients had refractory disease to HMA+Ven induction. The median time to achieve CR/CRi was 35 days (range, 22-230). Most CR/CRi patients (86%, n=18) continued consolidation therapy with HMA+Ven. Until changing the HMA+Ven regimen for any reason, the median number of HMA+Ven cycles received was 6 (range, 1-13). Only one patient received allogeneic stem cell transplant allo-HSCT after achieving remission and received gilteritinib maintenance therapy post-transplant. Six (29%) of the CR/CRi patients had relapse with a median time to relapse of 6 months (range, 1-10). The median duration of response was 13 months (95% CI: 6.71-19.28) (Figure 1A), and the median OS was 15 months (95% CI: 8.89-21.10) (Figure-1B). FLT3i (gilteritinib) was received in 6 patients (20%) post HMA+Ven; 2/6 received as maintenance therapy and 4/6 for relapse disease. In the univariate analysis for OS, diploid cytogenetics (p<0.0001) and non-high risk cytogenetics (p=0.04) were associated with significantly better OS (Figure-1D/E). AML sub-type (de novo vs sAML) (p=0.97), NPM1 mutation at diagnosis (p=0.39), FLT3m subtype (ITD vs TKD) (p=0.79) or allelic ratio at diagnosis (<0.5 vs >0.5) (p=0.89), and HMA sub-type (azacitidine vs decitabine) (p=0.43) were not significantly associated with difference in OS (Figure-1C/F/G/H, respectively). Conclusions: In summary, HMA+ Ven showed significant activity in treatment naïve FLT3m AML patients who were ineligible for intensive chemotherapy. HMA+Ven can be used as a backbone in combination with FLT3 inhibitor to improve outcome further and for successful transition to curative intent therapy such as allo-HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Results from a Phase I/II Study of the Triplet Combination of Azacitidine, Venetoclax and Gilteritinib for Patients with FLT3-Mutated Acute Myeloid Leukemia

Updated Results from a Phase I/II Study of the Triplet Combination of Azacitidine, Venetoclax and Gilteritinib for Patients with FLT3-Mutated Acute Myeloid Leukemia

FLT3-ITD Does Not Predict Inferior Prognosis Compared with FLT3-wild Type in Acute Myeloid Leukemia (AML) Patients Age ≥ 60 Years: A Retrospective Cohort Study

IntroductionFLT3 mutations are found in up to 30% of AML patients (pts), with 80% being internal tandem duplications (ITD). Based largely on data in younger pts from an era prior to the use of FLT3 inhibitors or hypomethylating agents (HMA) and venetoclax (Ven), FLT3-ITD mutated (FLT3-ITD+) AML is associated with higher relapse rates and decreased survival compared to FLT3 wild-type (WT) disease. Post hoc analysis from the VIALE-A trial demonstrated comparable outcomes between pts with FLT3-mutated vsFLT3-WT AML treated with HMA+Ven. Further, the AMLSG16-10 trial demonstrated improved survival among pts aged 60-70 with FLT-ITD+ AML treated with "7+3” and midostaurin compared to historical controls. To determine the clinical significance of FLT3-ITD+ in older adults with AML, we assessed the prevalence of FLT3-ITD mutations in pts with AML age ≥ 60 years treated at Dana Farber Cancer Institute (DFCI), evaluated whether FLT3-ITD+ is associated with an inferior prognosis in this age group and identified other specific characteristics affecting survival. Methods We included older (age ≥ 60) pts diagnosed with AML at DFCI between August 2015 and September 2021 and treated with: daunorubicin and cytarabine ("7+3") with or without a third drug; liposomal daunorubicin and cytarabine (CPX351); or HMA-based therapy (HMA+/-Ven). Groups were defined by presence (FLT3-ITD+) or absence (FLT3-ITD-) of FLT3-ITD mutation. We excluded pts in whom diagnostic molecular analysis was not performed, those who had acute promyelocytic leukemia or who received other treatments. Overall survival (OS) was evaluated by the Kaplan-Meier method. Cox regression models were fitted to assess the effect of FLT3-ITD mutations and additional covariates (age, prior myeloid disease, cytogenetics, molecular groups, treatment, and transplant as time-dependent covariate) on OS. Results Of the 380 pts, 53 (14%) were FLT3-ITD+ and 327 (85%) FLT3-ITD-. The median age was 69 years (range 60-89) and was similar between groups (68 [range 60-87] vs 70 [range (60-89], respectively, p=0.18). Intensive chemotherapy (IC) was used in 179 pts: 111 (62%) with "7+3", 20 (11%) with "7+3"+midostaurin (15 [47%] in FLT-ITD+ and 5 [3%] in FLT-ITD- in pts with FLT-TKD mutation), 16 (9%) with "7+3"+other non-FLT3 inhibitor investigational therapy and 32 (18%) with CPX351. HMA+Ven or HMA was used in 111 (29%) and 90 (24%) of pts, respectively. Baseline characteristics in FLT3-ITD+ and FLT3-ITD- pts differed by rates of normal karyotype, monosomal and/or complex karyotype (both p<0.001), and specific molecular co-mutations: (TP53 [p=0.032], secondary type mutations [0.045] and NPM1 [p=0.001]. Table). The median OS of the entire cohort was 13.6 months (CI 95% 11-16) and did not differ between pts with FLT3-ITD+vsFLT3-ITD- (median OS 15.4 [CI 95% 11-28] vs 13.1 [CI 95% 11-16], p=0.33). The median OS was similar between FLT3-ITD+vsFLT3-ITD- pts among those who received IC: 24.8 months (CI 95% 15-NA) vs 19.1 (CI 95% 13-30; p=0.43) and HMA-based-therapy: 9.33 months (CI 95% 6-NA) vs 9.9 (CI 95% 8-14; p=0.95), respectively (Figure). The addition of midostaurin could potentially ameliorate an adverse impact of a FLT3-ITD mutation. Thus, we conducted a sensitivity analysis in IC-treated pts who did not receive midostaurin, which demonstrated similar outcomes in FLT3-ITD+ ( n=17) vsFLT-ITD- (n=142): Median OS 15.4 months (CI 95% 4-NR) vs 17.5 (CI 95% 13-30), respectively, p=0.97. In a multivariable analysis ( MVA), FLT3-ITD status was not associated with OS (HR 1.1 [CI 95% 0.7-1.6] p=0.66). Factors associated with OS in the MVA were: treatment with HMA+Ven vs HMA (HR 0.62 [CI 95% 0.4-0.9], p=0.01), age ≥ 70 years (HR 1.03 [CI 95% 1.003-1.1], p=0.03), monosomal and/or complex karyotype vs normal karyotype (HR 1.67 [CI 95% 1.2-2.4], p=0.009) and TP53 mutations (HR 1.63 [CI 95% 1.1-2.3], p=0.005). Of note, treatment with intensive therapy vs HMA was not associated with OS (0.98 [CI 95% 0.7-1.5], p=0.93). Conclusion In older pts with AML, FLT3-ITD mutations were less common than reported in younger pts and were not associated with an adverse prognosis. Age ≥ 70 years, complex/ monosomal karyotype and presence of TP53 were associated with worse survival. Compared to HMA, therapy with HMA+Ven was associated with improved OS, while treatment with IC was not. The prognostic significance of a FLT3-ITD mutation should be re-evaluated in older pts with AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptomic-Based in silico Drug Screening Reveals Dual PI3K/mTOR Inhibitors Overcome Cabozantinib-Resistance of FLT3-ITD AML Cells

Background: Elucidating the mechanisms involved in tyrosine kinase inhibitor (TKI) resistance is critical for developing effective therapies in acute myeloid leukemia (AML). Cabozantinib (CBZ), an oral multi-target TKI, is well tolerated in AML patients and effectively inhibits the FLT3-mutated AML. We previously established CBZ-resistant MV4-11 cells (MV4-11-XR). However, the precise mechanisms of CBZ resistance are still not fully studied. Aims: To elucidate the molecular mechanism in MV4-11-XR and explore the potential therapeutic approaches to overcome CBZ-resistance. Materials and Methods: MV4-11-P and MV4-11-XR myeloid leukemia cell lines were used in this study. Cabozantinib (BMS-907351), omipalisib (GSK2126458), and gedatolisib (PF-05212384) were used for experiments. Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression was analyzed by immunoblotting. RNA-seq was performed using an Illumina NextSeq 500 platform. Sanger's sequencing and pyrosequencer were used to characterize the SNVs (single nucleotide variants). FusionCatcher was also used to explore potential fusion events related to resistance. Differentially expressed genes (DEGs) between MV4-11-P and MV4-11-XR were identified by EBseq. A threshold of p ≤ 0.05 was used to select the DEGs. Connectivity map was used to predict small molecules which had potentials to reverse enriched metabolic pathways in MV4-11-XR. The quantitative reverse transcription PCR (qRT-PCR) was performed by QuantStudio 3 Real-Time PCR Systems. The cellular ATP production rate was analyzed by the XFe 24 extracellular flux analyzer. Flow cytometry was used for cell cycle and apoptosis analysis. Results: We demonstrated that the anti-proliferative effect of CBZ on MV4-11-XR and parental MV4-11 (MV4-11-P). MV4-11-XR was significantly resistant to CBZ with IC50 of 1500 nM compared with MV4-11-P with IC50 of 9.5 nM. Subsequently, we performed RNA-seq to evaluate SNVs, indels, and enriched pathways in MV4-11-XR compared to MV4-11-P. We found 376 SNVs in MV4-11-XR. Of them, we noticed the emergence of FLT3 c.2503G>T (p.D835Y) mutation in MV4-11-XR. Pyrosequencing analysis confirmed that the frequency of mutant allele increased from 5 % in MV4-11-P to 99 % in MV4-11-XR. Immunoblotting confirmed the hyperactivation of FLT3 downstream signaling pathways in MV4-11-XR compared to MV4-11-P. FusionCatcher analysis indicated no novel fusion transcript related to CBZ resistance. Transcriptomic analysis revealed that a total of 1113 (366 upregulated and 725 downregulated) DEGs were detected in MV4-11-XR compared to MV4-11-P. Metascape analysis revealed that those up-regulated DEGs were enriched in "HALLMARK MTORC1 SIGNALING” and "HALLMARK HYPOXIA", indicating increased glycolysis in MV4-11-XR. qRT-PCR validated the overexpression of glycolytic genes including HIF1A, GLUT1, HK2, PKM2, LDHA, and PDK1 in MV4-11-XR. Real-time ATP rates analysis showed that MV4-11-XR had significantly increased rates of glycolytic ATP production. To explore small molecules that have the potential to reverse the metabolic phenotypes and overcome CBZ resistance, we performed a connectivity map analysis and predicted a PI3K inhibitor (wortmannin) and an mTOR inhibitor (sirolimus) as candidates. Therefore, we selected and evaluated the effects of gedatolisib and omipalisib, which have dual PI3K/mTOR inhibitory capacity, on MV4-11-XR. qRT-PCR showed that omipalisib significantly inhibited the expression of glycolytic genes in MV4-11-XR. Further experiments showed that gedatolisib and omipalisib significantly inhibited the proliferation of MV4-11-XR with IC50 of 28.33 nM and 37.56 nM, respectively. More importantly, gedatolisib and omipalisib significantly suppressed PI3K-AKT-mTOR signaling, promoted cell apoptosis, and induced cell cycle arrest in MV4-11-XR. Summary: In this study, we identified the emergence of FLT3 D835Y mutation and the upregulation of glycolysis in MV4-11-XR. As suggested by connectivity map analysis, we identified PI3K/mTOR dual inhibitor gedatolisib and omipalisib had potent anti-leukemic effects and could reverse the metabolic signature of CBZ-resistant cells. These findings highlight the potentials of PI3K/mTOR dual inhibitors as therapeutic molecules in AML to overcome TKI resistance.

The Application of the SORE6 Reporter in the Studies of Acute Myeloid Leukemia - a Novel Model for Disease Relapses

Most patients diagnosed with acute myeloid leukemia (AML) suffer from disease relapses within two years after the initial remission. Treatment options for relapsed AML are limited and the clinical outcome associated with this stage of disease is dismal. Partly due to the lack of experimental models, the biology underlying AML relapses is not well understood. We hypothesized that the cancer stem-like (CSL) cells, which represent a minority of the neoplastic cell population, are the major contributors of AML relapses. SORE6, a reporter designed to detect the expression and activity of inducible pluripotent stem cell factors, was used to identify/purify CSL cells in two FLT3-mutated AML cell lines, MOLM-13 and MV4-11. Both cell lines contained ~10% of SORE6+ cells at the steady state, as revealed by their expression of the reporter green fluorescence protein. Compared to SORE6- cells, SORE6+ cells had significantly more spheroid formation in the hanging drop assay (75 vs 35%, p=.006), and were significantly more resistant to Ara-C (inhibitory concentration at 50%, 60 vs 25 nM, p=.0005) and Venetoclax (2.6 vs 8.2 nM, p=.002) in the presence of low-dose Ara-C. By western blots, SORE6+ cells were found to express substantially higher levels of Myc/FLT3/pSTAT5 than SORE6- cells. Myc appeared to be a key driver of SORE6 activity, and the reporter activity was effectively suppressed by Myc pharmacologic inhibitors or shRNA. By subjecting a mixture of bar-coded SORE6- and SORE6+ MOLM-13 cells (in 9:1 ratio to mimic that at the steady state) to the lowest concentration of Ara-C that resulted in no detectable viability in the cell culture, we consistently found evidence of cell regeneration after 8-12 days of undetectable cell viability. Regeneration initially occurred slowly, and the cell growth went into a log phase after 1-2 weeks. Using PCR to detect the relative proportion of bar-coded SORE6- and SORE6+ cells, we found that the regenerated cell population contained a substantially higher proportion of SORE6+ cells, with 64% SORE6+ cells in the initial slow-growth phase. In comparison, the proportion of cells originating from SORE6+ cells increased to an average of 86% during the log-growth phase. Importantly, cells in the log-growth phase were found to have a significantly higher resistance to Ara-C and had significantly more spheroid formation in the hanging drop assay compared to untreated cells. To further validate the concept that CSL cells are the key contributors to the AML relapse, we collected pairs of initially diagnostic and relapsed bone marrow specimens derived from the same patients. By immunocytochemistry, we found that relapsed AML blasts were more frequently positive for Myc, a surrogate marker of SORE6 activity, compared to those in the pre-treatment samples. To conclude, using SORE6, we have developed a technically simple experimental model that capitulates some of the key features of AML relapses. Our model has provided direct evidence that CSL cells are key contributors to the disease relapse in AML. We believe that this in-vitro model is valuable in testing the efficacy of new therapeutic agents against AML, especially those designed to target the CSL cell population.

Molecular Characterization of Clinical Response in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome Patients Treated with Single Agent Emavusertib

Introduction Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are myeloid malignancies that exhibit a dynamic mutational landscape as the disease progresses. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of interleukin-1 receptor-associated kinase 4 (IRAK4), which is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Splicing factor mutations are frequently reported in AML and MDS and have been associated with disease progression and poor prognosis in relapsed/refractory (R/R) diseases. Emavusertib is a potent oral inhibitor of IRAK4 as well as Fms-like tyrosine kinase 3 (FLT3). The safety, clinical activity, and potential biomarkers of emavusertib in AML or high-risk MDS (HR-MDS) are investigated in the ongoing open-label, phase 1/2a TakeAim Leukemia trial (NCT04278768). Methods Eligible patients with R/R AML or HR-MDS were enrolled and treated with emavusertib. The exploratory objectives of the study are to assess the association between clinical activity and target-related biomarkers, gene expression signatures, and molecular subtypes. Bone marrow or peripheral blood were collected at the baseline and on treatment. To characterize the mutational profiles of enrolled patients and further define the targeted population, targeted next generation sequencing (NGS) of 68 genes was performed on genomic DNA from bone marrow or peripheral blood mononuclear cells. A minimum coverage of 100x, variant allele frequency (VAF) of >1%, and a VARSCAN-assigned variant p<0.001 were used as thresholds for single nucleotide variants (SNV) calling and presumed somatic driver mutations were identified as previously reported (Metzeler et al. Blood 2016). Mutations from patients’ molecular pathology reports were also documented. Results As of December 16, 2021, 49 patients have been treated with emavusertib monotherapy at one of dose levels of 200, 300, 400, or 500 mg BID, including 30 R/R AML and 19 R/R HR-MDS. The median number of prior anti-cancer therapies was 2 (range 1-5). This abstract is intended to add additional molecular findings to the patient subset presented in 2022 ASCO, of which 6 AML and 7 HR-MDS had splicing factor SF3B1 or U2AF1 mutations and 3 AML had FLT3 mutations, including 2 AML with both splicing factor and FLT3 mutations (hereafter referred to as "targeted mutations"). In the patients with targeted mutations, with available mutational profile data or molecular reports from sites, there were 6 responders: 1 CR, 1 CRh, and 4 mCR. In this responder population, the most common co-mutation was ASXL1. During emavusertib treatment, FLT3-mutated clones became undetectable in 2 of 3 (67%) FLT3-mutated AML patients. One of the 3 patients (33%) with a FLT3 mutation achieved CR and the remaining 2 patients achieved SD (1 with > 90% blast reduction). Additional gene mutations, such as ASXL1, RUNX1, CEBPA, ETNK1, ETV6, and SRSF2, became undetectable during emavusertib treatment. New mutations that emerged after ≥ 1 cycle of emavusertib treatment include NRAS, SETBP1, RUNX1, WT1, STAG2, KRAS, CCND2, DNMT3A, ASXL1, and NOTCH1. Analysis of genomic and transcriptomic profiles will be updated. Conclusion Emavusertib demonstrated anti-cancer activity in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1, SF3B1, or FLT3 mutations. Our preliminary NGS data is suggestive of molecular responses and disease-modifying activity of emavusertib. Patients with targeted mutations responded to emavusertib, even in the presence of co-mutations of ASXL1 that are typically associated with poor prognosis. We will continue to explore potential genomic biomarkers of emavusertib sensitivity and resistance

Streamline - Retrospective Cohort Study of FLT3-mutated Acute Myeloid Leukemia (AML): Real-World Treatment Patterns and Clinical Outcomes after First Relapse or Refractory (R/R) Diagnosis

Streamline - Retrospective Cohort Study of FLT3-mutated Acute Myeloid Leukemia (AML): Real-World Treatment Patterns and Clinical Outcomes after First Relapse or Refractory (R/R) Diagnosis

Gilteritinib activity in refractory or relapsed FLT3-mutated acute myeloid leukemia patients previously treated by intensive chemotherapy and midostaurin: a study from the French AML Intergroup ALFA/FILO.

The real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients. Among them, 140 received gilteritinib as single agent (cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (cohort C). The main differences in patient characteristics in this study compared to the ADMIRAL trial were ECOG ≥ 2 (83.6% vs. 16.6%), FLT3-TKD mutation (21.0% vs. 8.5%), primary induction failure (15.0% vs. 40.0%) and line of treatment (beyond 2nd in 37.1% vs. 0.0%). The rates of composite complete remission, excluding those that occurred after hematopoietic stem cell transplantation (HSCT), were similar at respectively 25.4% and 27.5% in cohorts B and C. Median overall survival (OS) for these two groups was also similar at respectively 6.4 and 7.8 months. Multivariate analyses for prognostic factors associated with OS identified female gender (HR 1.61), adverse cytogenetic risk (HR 2.52), and allogenic HSCT after gilteritinib (HR 0.13). Although these patients were more heavily pretreated, these real-world data reproduce the results of ADMIRAL and provide new insights into the course of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment who can benefit from treatment in an outpatient setting.

Therapeutic Targeting of FLT3 in Acute Myeloid Leukemia: Current Status and Novel Approaches.

FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients. The presence of FLT3-ITD (internal tandem duplication, 20-25%) mutation and, to a lesser extent, FLT3-TKD (tyrosine kinase domain, 5-10%) mutation is associated with poorer diagnosis and therapy response since the leukemic cells become hyperproliferative and resistant to apoptosis after continuous activation of FLT3 signaling. Targeting FLT3 has been the focus of many pre-clinical and clinical studies. Hence, many small-molecule FLT3 inhibitors (FLT3is) have been developed, some of which are approved such as midostaurin and gilteritinib to be used in different clinical settings, either in combination with chemotherapy or alone. However, many questions regarding the best treatment strategy remain to be answered. On the other hand, various FLT3-dependent and -independent resistance mechanisms could be evolved during FLT3i therapy which limit their clinical impact. Therefore, identifying molecular mechanisms of resistance and developing novel strategies to overcome this obstacle is a current interest in the field. In this review, recent studies of approved FLT3i and knowledge about major resistance mechanisms of clinically approved FLT3i's will be discussed together with novel treatment approaches such as designing novel FLT3i and dual FLT3i and combination strategies including approved FLT3i plus small-molecule agents targeting altered molecules in the resistant cells to abrogate resistance. Moreover, how to choose an appropriate FLT3i for the patients will be summarized based on what is currently known from available clinical data. In addition, strategies beyond FLT3i's including immunotherapeutics, small-molecule FLT3 degraders, and flavonoids will be summarized to highlight potential alternatives in FLT3-mutated AML therapy.

The Growing Role of the BH3 Mimetic Drug Venetoclax in the Therapy of Acute Myeloid Leukemia.

Despite recent progress, acute myeloid leukemia (AML) remains a disease associated with poor prognosis, particularly in older AML patients unfit to tolerate intensive chemotherapy treatment. The development and introduction in the therapy of Venetoclax (VEN), a potent BH3 mimetic targeting the antiapoptotic protein BCL-2, inducing apoptosis of leukemic cells, has shown to be a promising treatment for newly diagnosed, relapsed, and refractory AML patients ineligible for induction chemotherapy. Combination treatments using Ventoclax and a hypomethylating agent (azacitidine or decitabine) or low-intensity chemotherapy have shown in newly diagnosed patients variable response rates, with highly responsive patients with NPM1, IDH1-IDH2, TET2, and RUNX1 mutations and with scarcely responsive patients with FLT3, TP53 and ASXL1 mutations, complex karyotypes, and secondary AMLs. Patients with refractory/relapsing disease are less responsive to Venetoclax-based regimens. However, in the majority of patients, the responses have only a limited duration, and the development of resistance is frequently observed. Therefore, understanding the resistance mechanisms is crucial for developing new strategies and identifying rational drug combination regimens. In this context, two strategies seem to be promising: (i) triplet therapies based on the combined administration of Venetoclax, a hypomethylating agent (or low-dose chemotherapy), and an agent targeting a specific genetic alteration of leukemic cells (i.e., FLT3 inhibitors in FLT3-mutated AMLs) or an altered signaling pathway; (ii) combination therapies based on the administration of two BH3 mimetics (i.e., BCL-2 +MCL-1 mimetics) and a hypomethylating agent.

Rationale for Combining the BCL2 Inhibitor Venetoclax with the PI3K Inhibitor Bimiralisib in the Treatment of IDH2- and FLT3-Mutated Acute Myeloid Leukemia.

In October 2020, the FDA granted regular approval to venetoclax (ABT-199) in combination with hypomethylating agents for newly-diagnosed acute myeloid leukemia (AML) in adults 75 years or older, or in patients with comorbidities precluding intensive chemotherapy. The treatment response to venetoclax combination treatment, however, may be short-lived, and leukemia relapse is the major cause of treatment failure. Multiple studies have confirmed the upregulation of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family and the activation of intracellular signaling pathways associated with resistance to venetoclax. To improve treatment outcome, compounds targeting anti-apoptotic proteins and signaling pathways have been evaluated in combination with venetoclax. In this study, the BCL-XL inhibitor A1331852, MCL1-inhibitor S63845, dual PI3K-mTOR inhibitor bimiralisib (PQR309), BMI-1 inhibitor unesbulin (PTC596), MEK-inhibitor trametinib (GSK1120212), and STAT3 inhibitor C-188-9 were assessed as single agents and in combination with venetoclax, for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Enhanced cytotoxic effects were present in all combination treatments with venetoclax in AML cell lines and AML patient samples. Elevated in vitro efficacies were observed for the combination treatment of venetoclax with A1331852, S63845 and bimiralisib, with differing response markers for each combination. For the venetoclax and bimiralisib combination treatment, responders were enriched for IDH2 and FLT3 mutations, whereas non-responders were associated with PTPN11 mutations. The combination of PI3K/mTOR dual pathway inhibition with bimiralisib and BCL2 inhibition with venetoclax has emerged as a candidate treatment in IDH2- and FLT3-mutated AML.

Concomitant targeting of FLT3 and BTK overcomes FLT3 inhibitor resistance in acute myeloid leukemia through the inhibition of autophagy.

Strategies to overcome resistance to FMS-like tyrosine kinase 3 (FLT3)-targeted therapy in acute myeloid leukemia (AML) are urgently needed. We identified autophagy as one of the resistance mechanisms, induced by hypoxia and the bone marrow microenvironment via activation of Bruton tyrosine kinase (BTK). Suppressing autophagy/BTK sensitized FLT3- mutated AML to FLT3 inhibitor-induced apoptosis. Furthermore, co-targeting FLT3/BTK/aurora kinases with a novel multikinase inhibitor CG-806 (luxeptinib) induced profound apoptosis in FLT3-mutated AML by co-suppressing FLT3/BTK, antagonizing autophagy, and causing leukemia cell death in FLT3-wildtype AML by aurora kinase-mediated G2/M arrest and polyploidy, in addition to FLT3 inhibition. Thus, CG-806 exerted profound anti-leukemia activity against AML regardless of FLT3 mutation status. CG-806 also significantly reduced AML burden and extended survival in an in vivo patient-derived xenograft leukemia murine model of FLT3 inhibitor-resistant FLT3-ITD/TKD double-mutant primary AML. Taken together, these findings indicate that CG-806 has a unique mechanistic action and pre-clinical activity, which is presently undergoing clinical evaluation in both FLT3 wildtype and mutant AML.

Itraconazole-Induced Increases in Gilteritinib Exposure Are Mediated by CYP3A and OATP1B.

Gilteritinib, an FDA-approved tyrosine kinase inhibitor approved for the treatment of relapsed/refractory FLT3-mutated acute myeloid leukemia, is primarily eliminated via CYP3A4-mediated metabolism, a pathway that is sensitive to the co-administration of known CYP3A4 inhibitors, such as itraconazole. However, the precise mechanism by which itraconazole and other CYP3A-modulating drugs affect the absorption and disposition of gilteritinib remains unclear. In the present investigation, we demonstrate that pretreatment with itraconazole is associated with a significant increase in the systemic exposure to gilteritinib in mice, recapitulating the observed clinical drug–drug interaction. However, the plasma levels of gilteritinib were only modestly increased in CYP3A-deficient mice and not further influenced by itraconazole. Ensuing in vitro and in vivo studies revealed that gilteritinib is a transported substrate of OATP1B-type transporters, that gilteritinib exposure is increased in mice with OATP1B2 deficiency, and that the ability of itraconazole to inhibit OATP1B-type transport in vivo is contingent on its metabolism by CYP3A isoforms. These findings provide new insight into the pharmacokinetic properties of gilteritinib and into the molecular mechanisms underlying drug–drug interactions with itraconazole.

AML-241 Sequential Venetoclax and FLT3 Inhibitors in Combination With Hypomethylating Agents or Low-Dose Chemotherapy in Newly Diagnosed, Unfit, FLT3-Mutant Acute Myeloid Leukemia Patients

Targetable mutations such as FLT3, a receptor tyrosine kinase, are present in 30% of cases with acute myeloid leukemia (AML). Direct inhibitors of FLT3 are promising therapeutic agents such as midostaurin and gilteritinib. The use of FLT3 inhibitors as single agents remains to be limited with short-lived responses. For treatment-naïve AML patients with FLT3 mutation, the standard treatment entails induction with intensive chemotherapy in physically fit or younger patients and in combination with an FLT3 inhibitor. There is no approved targeted therapy option for treatment-naïve patients with AML harboring an FLT3 mutation ineligible for intensive therapy. To assess the efficacy and feasibility of sequential administration of venetoclax, a selective inhibitor of Bcl-2, and midostaurin in addition to induction chemotherapy in unfit newly diagnosed FLT3-mutant AML patients. This is a case series reviewing the clinical data of four consecutive patients with FLT3-mutant AML, ineligible for standard induction chemotherapy, who received induction treatment consisting of low-dose chemotherapy or hypomethylating agents (HMA) with the sequential use of venetoclax for 10 days followed by midostaurin for 14 days, treated at the American University of Beirut Medical Center. Between November 2021 and February 2022, four patients were included in our study. The median age was 68 years (range 66-70). All patients had FLT3 mutation (three with ITD, one with TKD), and three patients had concomitant NPM1 mutation. One patient had trisomy 8 and another patient had deletion 7 on cytogenetics. All patients achieved complete remission on day 21 and day 28. One patient with underlying myelodysplastic syndrome remained to have detectable FLT3 with deletion 7 at day 28 evaluation and had the longest time for absolute neutrophil count (ANC) and platelet count recovery. The median number of days for ANC and platelet count recovery was 40 days (range 27-59) and 34 days (24-not reached) respectively. The main toxicity was febrile neutropenia in 3 out of 4 patients. The sequential use of venetoclax followed by midostaurin in combination with low dose chemotherapy or HMA for newly diagnosed AML, FLT3-mutant patients who are unfit for intensive chemotherapy seems to be a promising approach with an acceptable toxicity profile.