FLT3 In Acute Myeloid Leukemia Research Articles

Investigating the Impact of Smoking Traditional and E-Cigarettes on Acute Myeloid Leukemia Progression

Acute myeloid leukemia (AML) is a highly heterogeneous disease with a variable prognosis. The exact mediators of AML are not fully understood but there are several factors that may contribute to the development of the disease. These can be split into genetic and non-genetic causes. cytogenetic abnormalities have been identified such as FLT 3 gene which frequently mutated in AML and associated with a poor prognosis. As for non-genetic causes: prior exposure to chemotherapy or radiation therapy have been implicated. Lately cigarette smoking in AML has been shown to have poor survival outcome for AML patients relative to non-smokers. Cigarette smoke contains many compounds, some of which have been associated with increased risk for AML. Benzene, radioactive components, and possibly other carcinogens are present in tobacco and tobacco smoke which lead to production of chromosomal defects. Cigarette smoke exposure globally alters DNA methylation in blood cells and these changes can persist for decades. All commercial tobacco products, including smokeless tobacco, cigarette smoke and E cigarettes contain tobacco-specific nitrosamines such asN′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK and NNN promotes tumor growth by enhancing and deregulating cell proliferation, survival, migration, and invasion, thereby creating a microenvironment for tumor growth. This calls for a need for to understand the molecular changes in AML especially when exposed to agents that can impact survival. Therefore, we hypothesize when FLT-3 AML model is exposed to smoking metabolites such as cigarettes smoking condensate (CSC), NNN, NNK and E cigarettes can lead to increased leukemia burden. To test this in vitro CSC treatment of AML cells was used to evaluate how chemicals from cigarette smoke can impact AML cells without the high heat combustion from cigarettes. NNN and NNK treatment were separately used for treatment of AML cells. Two FLT-3 AML cell lines used (MOLM-13 and MV-4-11) and non-FLT-3 AML cell line (OCI-AML3) as control.Cells were treated in vitro with 10 ug/ml cigarette smoke condensate (CSC) or 1uM NNN or 1 uM NNK or vehicle (DMSO). Viability and cell proliferation were measured with each passage every 48-72 hours. There was no difference in cell proliferation and viability with CSC, NNN or NNK exposure. This suggests possibly that microenvironment is needed to evaluate the impact of smoking on AML progression. To test this in vivo, luciferase tagged MOLM13 cells were treated in vitro with 10ug/ml cigarette smoke condensate (CSC) or vehicle DMSO for two weeks then injected into mice. Mice injected with MOLM 13 cells treated with CSC had significantly more leukemia burden than control. Similar findings were replicated in MV-4-11 mouse model. Next, we wanted to evaluate if this increased leukemia burden leads to DNA methylation changes. We Found 8 genes to be significantly hypermethylated from the CSC two-week in vitro treatment. This was also found in the vivo experiment where mice were exposed to Cigarette smoking in vivo. (Table 1) This study shows that CSC treated AML cell lines can lead to increase leukemia burden in vivo but not in vitro. There are difference in DNA methylation between AML cells lines exposed to CSC compared to control. Our future directions will include evaluate leukemia burden in FLT-3 AML mouse model exposed to NNN, NNK and E- cigarettes Study ROS changes, protein expression and DNA methylation impacted by smoking.

The FLT3-like Gene Expression Signature Predicts Response to Quizartinib in Wild-Type FLT3 Acute Myeloid Leukemia: An Analysis of the Pethema Quiwi Trial

Background The identification of predictive biomarkers is crucial for guiding treatment decisions in acute myeloid leukemia (AML). Previously, we identified a FLT3-like gene expression signature in FLT3 wild-type AML patients, which clustered a variable proportion of FLT3 wild-type patients with FLT3-ITD and TKD mutated patients. The QUIWI trial was a randomized, placebo-controlled, phase II study preliminary showing a significant increase in overall survival (OS) in wild-type FLT3 AML patients treated with the FLT3 inhibitor quizartinib (Quiza) plus standard chemotherapy. This preplanned correlative study was designed to assess the value of the FLT3-like signature to predict responses to Quiza. Methods We performed RNA sequencing (RNAseq) analysis on a subset of patients from the clinical trial. RNA was extracted using standard methods, followed by assessment of nucleic acid integrity (TapeStation) and quantification (Qubit). Total mRNA sequencing was performed using polyA RNAseq with TruSeq technology. A total of 206 adequate samples from bone marrow and peripheral blood were sequenced (161 from FLT3-ITD negative enrolled in QUIWI; and 55 from FLT3-ITD positive patients who were screen failure of the QUIWI trial by this reason). The sequences were aligned to the GRCh37 reference genome using the Hisat algorithm. Gene expression quantification was performed using the Bioconductor workflow, and gene expression estimates (FPKM) were obtained. The gene expression estimates were log2 normalized. Finally, those genes mapping to the original FLT3-like signature (595 genes) were selected for downstream analysis. Clustering was based on the hierarchical method, using standard euclidean distance metrics. OS was defined as time from start of screening to death. Event-free survival (EFS) was defined as time from randomization to failure to achieve CR/CRi after 1 or 2 cycles, death in CR/CRi, or relapse (whichever occurred the first). Relapse-free survival was defined as time from randomization to disease relapse or death by any cause. Results Among the total 206 patients, a cluster of 54.37% (N=112) was enriched in FLT3-mutant cases (71.11% of cases, Figure 1A). This subgroup comprised 49.67% of wild-type FLT3 cases (N=80), hereafter FLT3-like patients. In the group of not FLT3-like patients, no differences were identified between the placebo and Quiza group in the total number of deaths (Fisher's p-value, 0.63), EFS (cox p-value, 0.83; HR 1.07 [0.56-2.06]), RFS (cox p-value 0.76; HR 0.88 [0.38-2.01]) and OS (cox p-value, 0.62; HR 1.22 [0.55-2.67]). Among FLT3-like patients, significant differences were identified in the total number of deaths (Fisher's p-value, 0.004), EFS (cox p-value, 0.009; HR 0.45 [0.25-0.82], RFS (cox p-value 0.01, HR 0.37 [0.18-0.79]) and OS (cox p-value 0.01, HR, 0.41 [0.20-0.84]) ( Figure 1B). No statistically significant association was observed between the FLT3-like pattern and the ELN-17 classification: 30.4% were low risk, 40.5% intermediate risk, 29.1% high risk. Conclusion The FLT3-like gene expression signature successfully identified a subset of patients who derived the most benefit from Quiz, while patients without the FLT3-like signature did not demonstrate a benefit compared with placebo. These findings support the use of the FLT3-like signature as a potential biomarker to identify those wild-type FLT3 AML patients who may benefit from Quiz, providing a valuable insight for personalized treatment in AML.

FMS-like tyrosine kinase 3 internal tandem duplication mutation in patients with acute myeloid leukemia in Kurdistan region/Iraq

BACKGROUND: Mutations in the FLT3 gene are very common in acute myeloid leukemia (AML). These mutations are linked to a high chance of the disease relapse and decrease overall survival. This study aimed to investigate this mutation in AML cases and to correlate it with the clinicopathological presentation and their response to induction therapy. OBJECTIVES: This study aimed to investigate this mutation in AML cases and to correlate it with the clinicopathological presentation and their response to induction therapy. METHODS: This study was done on 63 AML cases; for each patient, the clinical presentation and the hematological lab parameters were recorded. The results of FLT3 internal tandem duplication (ITD) mutation were recorded and the mutation was detected by conventional polymerase chain reaction technology. Postinduction assessment was recorded for each patient. RESULTS: The mean age of the studied group was 36.06 (22.42), with a male to female ratio of 1:1.6. Out of 63 AML patients, 16 (25.4%) had FLT3-ITD mutation. The highest incidence of the mutation was found among the age group of 10–19 years (40%). The highest incidence of FLT3 mutation was among M3 (45.5%). The blast count was significantly higher in patients with than without the mutation (P = 0.04). The remission rate was significantly lower in FLT3 AML patients than in those lacking the mutation (P = 0.45). CONCLUSION: FLT3-ITD mutation was common in our AML patients. This mutation was associated with significantly higher counts of blast and poorer response to induction therapy; thus, it is considered one of the poor prognostic factors.

Prognostic Impact of Persistent Measurable Residual Disease in Patients with Acute Myeloid Leukemia FLT3+ Treated with Intensive Chemotherapy Plus Midostaurin - a Retrospective Study of the Polish Adult Leukemia Group (PALG)

Background: Measurable residual disease (MRD) is detected during morphological remission after standard chemotherapy in acute myeloid leukemia (AML) patients (pts). It is a strong prognostic factor for relapse and shorter overall survival (OS). Immunophenotyping by multiparameter flow cytometry (MFC) is currently the most commonly used method in AML pts. There is still a lack of studies evaluating MRD in specific AML populations in the era of targeted therapies, including thyrosine kinase inhibitors. A retrospective study in adult pts with a newly diagnosed AML FLT3+ treated with intensive chemotherapy plus midostaurin has been performed to evaluate the prognostic impact of positive MRD (MRD+) on RFS and OS. Objectives: To assess the prognostic impact of MRD status during the clinical course of AML FLT3+ on RFS and OS. Patients and methods: Seventy three pts with AML FLT3 were treated in Polish hematological centers between 2018 and 2021. Bone marrow aspiration was performed after induction and consolidation treatment to assess response and MRD by MFC. MRD+ was defined as ≥ 0.1% abnormal cells within mononucleated cells by MFC with diagnostic leukemia-associated immunophenotype (LAIP) and different from normal (DfN) aberrant immunophenotype approaches. Results: The median age of the pts was 55 (range: 18-74). Induction regimen was DA "3+7" in 50 (70%), DA-90 in 8 (11%), DAC in 11 (15%), other in 2 (4%) pts. Fifty seven (78%) pts achieved first composite complete remission (CRc=CR+CRi) after 1 or 2 courses of induction, 12 (17%) had primary refractory disease (PRD) and 2 (3%) pts died in aplasia. Median number of consolidation courses was 2 (range: 1-4). Fifty one (89%) pts with CR1 fulfill the criteria for MRD analysis after 1 or 2 courses of induction and 34 (48%) pts after consolidations. Thirty four pts had 2 timepoints evaluation: after induction and at the end of consolidation courses. MRD+ was reported in 35 (68%) of pts after induction (median 0.5; IQR 0.25-0.85) and in 19 (52%) of pts at the end of consolidations (median 0.64; IQR 0.22-1.99). Analysis of 4 pts groups was performed: Group 1.: MRD+ after induction and at the end of consolidations (persistent MRD+)(n=16; 47%); Group 2.: MRD+ after induction with conversion to MRD- at the end of consolidations (n=6; 18%); Group 3.: MRD- after induction with conversion to MRD+ at the end of consolidations (n=2; 6%); Group 4.: MRD- after induction and at the end of consolidations (persistent MRD-)(n=10; 29%); Baseline clinical characteristics were similar between 4 groups regarding age, FLT3-ITD and FLT3-TKD frequency, FLT3-ITD allelic ratio (AR), WBC, PLT, peripheral blood and bone marrow blast percentage, AML type: de novovs sAML vs tAML (p-values > 0.05). The probability of 1- year RFS was significantly lower in pts with persistent MRD+ (group 1.) vs group 2. vs 3. vs 4. (27% vs 83% vs 50% vs 78%; p=0.034, respectively). Relapse rate after 1 year was in group 1. vs 2. vs 3. vs 4.: 73% vs 16% vs 50% vs 20% (p=0.015), respectively. The shorter OS, but insignificant, was noted in group 1. vs 2. vs 3. vs 4. (p=0.49). A univariate analysis revealed that allo-HSCT (hazard ratio [HR] 0.345; 95% confidence interval (CI), 0.152-0.784; p=0.011) and male sex (HR, 0.433; 95% CI, 0.217-0.861; p=0.017) are predictors for longer OS. MRD- at the end of consolidations (HR, 0.211; 95% CI, 0.066-0.669; p=0.008), persistent MRD- (HR, 0.252; 95% CI, 0.070-0.909; p=0.035), allo-HSCT (HR, 0.416; 95% CI, 0.183-0.947; p=0.0368) are predictors for longer RFS. However, older age (HR, 1.049; 95% CI, 1.018-1.081; p=0.0017) and higher WBC (HR, 1.005; 95% CI, 1.002-1.008; p=0.0022) are predictors for shorter RFS. Conclusions: We observed high rate of CR1 MRD+ AML FLT3 pts after induction (68%) and at the end of consolidations (52%). Significantly lower RFS and higher relapse rate, but not OS, were noted in pts with persistent MRD+ in comparison to pts in other MRD groups. Longer follow-up period and larger studies are necessary to confirm our preliminary findings.

Elucidating the Mechanism of Action (MOA) of Navtemadlin, an MDM2 Inhibitor, and Its Synergy with Gilteritinib in Myeloid Malignancies

Introduction: Murine double minute 2 (MDM2) is the primary negative regulator of the tumor suppressor protein, p53. Navtemadlin (KRT-232), a potent, selective, orally available MDM2 inhibitor, is being evaluated in the phase 3 BOREAS trial of patients with relapsed/refractory myelofibrosis (MF; NCT03662126) and in phase 1b/2 trials in various tumor types. In preclinical studies, navtemadlin has overcome MDM2 dysregulation and has restored p53 activity to drive apoptosis in TP53WT malignancies. The primary determinant of tumor cell sensitivity to navtemadlin is TP53 mutation status. Because MDM2 is often overexpressed and p53 mutations are less common in treatment-naïve acute myeloid leukemia (AML) and MF, navtemadlin represents a rational therapeutic candidate for these malignancies (Prokocimer et al. Blood. 2017). The present study aims to elucidate the effects of navtemadlin on proteins downstream of p53 and synergy of navtemadlin with the FLT3 inhibitor gilteritinib in myeloid malignancies. Methods: Cell viability was evaluated in three TP53WT myeloid cell lines, MOLM-13 (FLT3 AML), MV-4-11, and UKE-1, and one TP53MUT myeloid cell line, HEL-92, treated with 1-10 µM navtemadlin for 72 h. Growth inhibition was determined using the ATP-based Cell Titer Glo assay. The treatment effect on cell cycle progression, BCL-2 family proteins, and apoptosis was determined by flow cytometry. The effects of navtemadlin (0.5-1 µM) + gilteritinib (0.5-1 µM) were assessed in MOLM-13 and MV-4-11 (TP53WT and TP53MUT clones) and visualized using Combenefit. A MOLM-13 TP53WT mouse xenograft model was used to assess in vivo activity. Engrafted mice were randomly assigned to one of five treatment groups (n=10 each): vehicle control, 30 mg/kg gilteritinib, 50 mg/kg navtemadlin, 50 mg/kg navtemadlin + 30 mg/kg gilteritinib, or 100 mg/kg navtemadlin. Mice were treated orally once a day until the mice met early removal criteria or the study was terminated. Survival was measured by the percentage of mice in each group that remained alive at the end of the study. Results: Navtemadlin exposure led to dose-dependent growth inhibition in the TP53WT myeloid cell lines MOLM13, MV-4-11, and UKE-1 with an EC90 of 2-6 µM. Conversely, no growth inhibition was observed in the TP53MUT myeloid cell line HEL-92, up to 10 µM of navtemadlin. Assessment of cell cycle progression following navtemadlin exposure showed the induction of threshold-dependent apoptosis in the TP53WT myeloid cell lines, but not in the TP53MUT myeloid cell line. The threshold at which a switch from p21-mediated cell-cycle arrest to p53-directed apoptosis occurs is illustrated in Figure 1a. Navtemadlin-induced apoptosis was indicated by a release of cytochrome c and increase in processed caspase-3. Clinically, a steady-state Cmax of 2-6 µM is achieved at therapeutically active and relevant doses of navtemadlin ≥180 mg given once a day on a 5-day or 7-day schedule in clinical trials, leading to maximum tumor cell death. To gain better insight into the contribution of BCL-2 family members alterations as an MOA for navtemadlin-induced apoptosis, expression levels of BCL-2 family members were evaluated in TP53WT myeloid cell lines. Navtemadlin increased levels of the effector pro-apoptotic proteins Bak, Bax, and Bim in leukemia cells. Synergy was observed in vitro for navtemadlin + gilteritinib in TP53WT, but not TP53MUT myeloid cell lines. Treatment with 100 mg/kg navtemadlin monotherapy and 50 mg/kg navtemadlin + gilteritinib combination led to prolonged survival in mice harboring MOLM-13 TP53WT xenografts, with all mice still alive at day 113 when the study was terminated (Figure 1b). In contrast, median survival times for the vehicle, 50 mg/kg navtemadlin monotherapy, and gilteritinib monotherapy groups were 18, 53, and 65 days, respectively. Conclusion: Navtemadlin induces potent, threshold-dependent apoptosis in cell culture models of TP53WT myeloid malignancies at clinically relevant concentrations. Navtemadlin-induced p53 activity initiates apoptosis by activating the caspase cascade via cytochrome c release. Navtemadlin was shown to induce the expression of pro-apoptotic BCL-2 family proteins, key mediators in driving cell death. These pro-apoptotic effects translate to robust antitumor activity in xenograft models of TP53WT myeloid malignancies, with the navtemadlin + gilteritinib combination demonstrating synergy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of Emergent FLT3 Mutations (FLT3m) at Relapse in Patients (Pts) with Wild-Type FLT3 Acute Myeloid Leukemia (AML)

Background:FLT3 mutations (FLT3m) are detected in approximately 30% of newly diagnosed (ND) pts with AML and are associated with poor prognosis. FLT3m can also emerge in relapse in pts with previously wild-type FLT3 AML; however, their frequency and clinical implications are not well studied. Our objective was to identify pts who relapsed with emergent FLT3m and compare their clinical and pathological characteristics to those who relapse without FLT3m. Methods: We identified 686 pts with ND AML treated at our institution between 12/2005 and 01/2020, who achieved complete remission (CR) / CR with incomplete count recovery (CRi) and subsequent relapse of AML. Pts with available FLT3 status (ITD and TKD) at diagnosis and within 4 weeks of first relapse were included (N=300). A multiplex fluorescent-based PCR analysis followed by capillary electrophoresis for detection of FLT3 ITD and/or TKD was performed on DNA isolated from BM aspirate samples. Next-generation sequencing (NGS) at baseline was performed using the Illumina MiSeq platform with multigene panels (either 28-gene, 53-gene, or 81-gene) interrogating genes recurrently mutated in myeloid neoplasms. Results: Among 300 pts who achieved CR/CRi after induction and eventually relapsed, 216 (72%) had FLT3 wild-type (WT) AML and 84 (28%) had FLT3m AML at diagnosis. Baseline clinical features are shown in Table 1. 43 (14%) pts had a change of FLT3 status between baseline and relapse: 30 pts had FLT3m at baseline that wasn't detected at relapse, and 13 pts with FLT3 WT AML at baseline experienced relapse with FLT3m AML. The median remission duration (until 1st relapse) for the entire study cohort pts withFLT3 WT and pts with FLT3m AML was 7.8 months (range, 0.2-147.2), 9 months (0.2-147.2), and 6.1 months (range, 0.5-79.6), respectively. FLT3 wild-type at Diagnosis Among 216 pts with FLT3 WT AML at baseline, 203 (94%) and 13 (6%) experienced relapse with FLT3 wild-type and FLT3m AML, respectively. Comparison of baseline molecular features of FLT3 WT and FLT3m cohorts showed that pts who had relapsed AML with a positive FLT3m had more frequent concomitant NRAS mutations compared with pts who had FLT3 WT AML at relapse (60% vs 5%; p=0.003). No significant difference was seen in the frequency of TP53 (49% vs. 20%; p=0.36), NPM1 (15% vs. 40%; p=0.19), DNMT3A (16% vs. 40%; p=0.21), KRAS (5% vs. 0%; p=1), or PTPN11 (3% v. 0%; p=1) mutations between the two cohorts, respectively. Among 13 pts with AML with emergent FLT3m, 12 had diploid cytogenetics at diagnosis, but only 6 maintained the diploid status at relapse. The remaining 6 pts developed karyotypic clonal evolution at relapse (complex, n=1; KMT2A rearrangement, n=1; trisomy 8, n=1; other alterations, n=3). We had paired baseline and relapse NGS performed in 5 of 13 pts with relapsed AML, among these AMLs acquired new mutations. Nine newly acquired mutations were detected in the following genes: NPM1 (n=1), KRAS (n=1), RUNX1 (n=1), IDH2 (n=1), DNMT3A (n=1), ZRSR2 (n=1), WT1 (n=2), and STAG2 (n=1). Two of 5 pts had AML with no mutations at baseline, and 1 pt had AML with the same mutation profile at baseline and relapse. The median OS after relapse was 5.3 months and 5.5 months in pts with WT vs. FLT3m AML, respectively (P=0.29) (Figure 1). FLT3 mutant at diagnosis Among 84 pts with FLT3m AML at diagnosis, 52 (62%) and 32 (38%) pts experienced relapse with FLT3m and WT AML, respectively. Comparison of baseline molecular features of FLT3 WT and FLT3m AML showed a trend towards a higher frequency of NPM1 (77% vs. 47%; p=0.09) and DNMT3A mutations (55% vs. 21%; p=0.08) in patients who experienced relapse with FLT3m AML compared with FLT3 WT AML. No difference in the frequency TP53 (0% vs. 23%; p=0.13), KRAS (0% vs. 14%; p=0.27), NRAS (14% vs. 9%; p=0.63) and PTPN11 (7% vs. 14%; p=1) mutations was observed among both cohorts, respectively. The median OS after relapse was 8.1 months vs. 3.9 months in pts with FLT3 WT vs. FLT3m AML, respectively (p=0.005) (Figure 2). Conclusion: Our data suggest that FLT3m are more likely to emerge at AML relapse with concomitant NRAS mutations and indicate that FLT3m testing should be performed at relapse, irrespective of FLT3status at diagnosis. It is common for the FLT3 status to change, either from mutant to WT or vice versa. FLT3 status has immediate and clinically important implications for selection of appropriate therapeutic agents in the setting of salvage therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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

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

S129: TAKEAIM LEUKEMIA- A PHASE 1/2A STUDY OF THE IRAK4 INHIBITOR EMAVUSERTIB (CA-4948) AS MONOTHERAPY OR IN COMBINATION WITH AZACITIDINE OR VENETOCLAX IN RELAPSED/REFRACTORY AML OR MDS

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Aims: Assessment of safety, clinical activity, and Recommended Phase 2 Dose (RP2D) of emavusertib as monotherapy or in combination with azacitidine (AZA) or venetoclax (VEN). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with emavusertib monotherapy. Phase 1b includes 2 arms of combination therapy: emavusertib + AZA and emavusertib + VEN. The primary objectives of this study are to assess the safety, clinical activity, and identify the RP2D of emavusertib as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for emavusertib monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of emavusertib were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to emavusertib. Summary/Conclusion: Emavusertib is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting emavusertib may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing.

Phase 1/2a study of the IRAK4 inhibitor CA-4948 as monotherapy or in combination with azacitidine or venetoclax in patients with relapsed/refractory (R/R) acute myeloid leukemia or lyelodysplastic syndrome.

7016 Background: CA-4948 is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) and FLT3. IRAK4 is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of IRAK4 and have been associated with disease progression and poor prognosis of high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML). Methods: This is an open-label, phase 1/2a dose escalation and cohort expansion trial (NCT04278768). In phase 1 Dose Escalation, patients with R/R AML or HR-MDS are treated with CA-4948 monotherapy. Phase 1b includes 2 arms of combination therapy: CA-4948 + azacitidine (AZA) and CA-4948 + venetoclax (VEN). The primary objectives of this study are to assess the safety, clinical activity, and identify the Recommended Phase 2 Dose (RP2D) of CA-4948 as monotherapy or in combination with AZA or VEN in R/R AML or HR-MDS. The Phase 2a Dose Expansion includes patients for CA-4948 monotherapy: R/R AML with FLT3 mutation, or AML and HR-MDS R/R to HMA with U2AF1 or SF3B1 mutations. Results: As of December 16th, 2021, 49 patients have been treated in the phase 1 portion, of whom 43 started by September 30th, allowing 2 on-study disease assessments. The median number of prior therapies was 2 (range 1-5). Four monotherapy dose levels of CA-4948 were tested (200 to 500 mg orally BID). No dose-limiting toxicities were observed at 200 mg and 300 mg BID. No Grade 4 or 5 treatment-related AEs (TRAEs) were reported, and all the TRAEs were manageable. Reversible, manageable Grade 3 rhabdomyolysis occurred in 1/26 (4%) patients at 300 mg BID, 2/17 (12%) at 400 mg BID, and 1/3 (33%) at 500 mg BID. RP2D was determined as 300 mg BID. Of 43 patients starting before Sept 30th, 2021, 14 had SF3B1, U2AF1 or FLT3 mutations and demonstrated more promising efficacy. In the 5 evaluable AML patients with spliceosome mutations, 40% reached CR/CRh (1 CR, 1 CRh), both with study duration >6 months. In the 7 spliceosome-mutated HR-MDS patients, 57% reached marrow CR, including 1 with RBC transfusion independence and 1 proceeding to HSCT. One of the three FLT3-mutated AML reached CR, and 2 became FLT3-negative. Among the 29 patients without SF3B1/U2AF1/FLT3 mutations, 1 reached CR and 2 PR. Phase 1b and Phase 2a are ongoing. RNA-seq on selected samples showed decrease in relative expression of IRAK4-long isoforms with response to CA-4948. Conclusions: CA-4948 is well tolerated and effective in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1/SF3B1/FLT3 mutations. No dose-limiting myelosuppression was reported, suggesting CA-4948 may be a candidate for combination therapy. Accrual of Phases 1b and 2a is ongoing. Clinical trial information: 04278768.

Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML.The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations.Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models.We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1.We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A).To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B).We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C).In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D).In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E).Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers.Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. [Display omitted] DisclosuresStahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Quizartinib (Quiz) with Decitabine (DAC) and Venetoclax (VEN) Is Highly Active in Patients (pts) with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) - RAS/MAPK Mutations Continue to Drive Primary and Secondary Resistance

Quizartinib (Quiz) with Decitabine (DAC) and Venetoclax (VEN) Is Highly Active in Patients (pts) with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) - RAS/MAPK Mutations Continue to Drive Primary and Secondary Resistance

Efficacy Analysis of Different FLT3 Inhibitors in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome Patients - a Systematic Review and Meta-Analysis

Background: Mutations in FMS like tyrosine kinase gene 3 (FLT3) are the most common genetic aberrations in acute myeloid leukemia (AML). Internal tandem duplications (ITD) are the most frequent FLT3 mutations, occurring in 20-30% of patients (pts) with newly diagnosed AML, and a minority are tyrosine kinase domain mutations. FLT3 mutation confers adverse prognosis in pts treated with standard chemotherapy (chemo) alone, and relapse risk is also higher compared to FLT3-wild type AML (HR-1.75). Studies of FLT3 inhibitors as monotherapy or in combination with chemo in the treatment of pts with FLT3-ITD relapsed/refractory (R/R) AML are associated with mixed results. No evidence synthesis has yet focused on the effect of FLT3 inhibitor alone in the treatment of R/R AML and high-risk myelodysplastic syndrome (HR-MDS). Hence, we analyzed the efficacy of various FLT3 inhibitors from different clinical trials used as monotherapy to treat pts with R/R AML and HR-MDS.Methods: We conducted a systematic review and meta-analysis of clinical trials of FLT3 inhibitors for pts with R/R AML and HR-MDS. We searched EMBASE and PubMed for clinical trials published between 1/1/2000 and 5/26/2021 using a combination of keywords and subject headings related to FLT3 inhibitors and AML. Titles/abstracts and full texts were screened by two independent reviewers, with conflicts arbitrated by a third reviewer. Studies were included if they were (1) full-length published journal articles that (2) reported the results of single-arm or double-arm phase I/II/III clinical R/R AML or HR-MDS. Outcomes of interest were composite response rate (CRc=complete response + complete response with incomplete count recovery) and overall response rate (ORR).Results: Thirty studies were included after the application of inclusion criteria (Figure 1). Two were excluded due to inadequate representation of pts with R/R AML and outcomes reported for stem cell transplant following FLT3 inhibitor use, respectively. Hence, 28 studies were included in the meta-analysis, with a total of 1927 pts. Quizartinib and sorafenib were the most frequently studied inhibitors (6 and 5 studies, respectively, Table 1). Heterogeneity testing was performed using Cochran's Q test and I 2 values. The Cochran's Q test p-value was less than 0.10 (p<0.01), and the I 2 value was more than 50% (I 2=87%), indicating the presence of heterogeneity. Thus, random-effects models were used. Asymmetry test was performed using Egger's linear regression test, which suggested the presence of publication bias (p=0.001; Figure 2). Thus, the trim-and-fill method was used to adjust for publication bias. Pooled ORR was 53% (95% CI, 43-63%) (Figure 2). Similarly, pooled CRc was 34% (95% CI, 26-44%, Figure 3). Pooled CRc and ORR were higher in studies involving quizartinib (40% and 61%, respectively) and gilteritinib (40% and 52%, respectively, Table 1). The CRc rates seen with FLT3 inhibitors are higher than the historical comparison of pts with FLT3-mutated-R/R AML treated with chemo only (CRc ~15%). This meta-analysis shows that FLT3 inhibitors as monotherapy leads to meaningful clinical responses in pts with FLT3-mutated-R/R AML.Conclusion: Most FLT3 inhibitors are effective as monotherapy for the treatment of pts with FLT3-mutated-R/R AML. Pooled response rates are notably higher in studies involving second-generation FLT3 inhibitors, particularly quizartinib and gilteritinib. Though not conclusive, the efficacy spectrum of various FLT3 inhibitors in the R/R setting could help design future studies and guide appropriate treatment selection; however, further validation is needed. Prospective clinical trials are required to compare the effectiveness of newer generation FLT3 inhibitors in pts with FLT3-mutated-R/R AML. [Display omitted] DisclosuresMaciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Balasubramanian: Servier Pharmaceuticals: Research Funding.

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs).Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape.Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis.Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide Reprograms the Bone-Marrow Microenvironment to Sensitize Minimal Residual Disease in Acute Myeloid Leukemia

Eliminating minimal residual disease (MRD) in order to prevent relapse is an unmet need in acute myeloid leukemia (AML). One of the success stories is acute promyelocytic leukemia (APL), a unique subtype of AML. The use of arsenic trioxide (ATO) in combination with all-trans retinoic acid (ATRA) has essentially eliminated disease relapse (<1% ATRA+ATO vs. ~18% ATRA+ chemotherapy - NEJM (2013), 369(2), 111-121). This is even more impressive as neither agent alone results in significant cure of patients with APL. In fact, single agent ATRA induces remission without cure due to bone marrow stroma expression of CYP26 enzymes (PloS one (2015), 10(6), e0127790), which are induced by ATRA, inactivate retinoids and protect APL MRD (Leukemia (2020), 34(11), 3077-3081). While ATO may synergize with ATRA by restoring PML nuclear bodies (Nature medicine (2014), 20(2), 167-174) and rescuing RAR-α dependent transcription (Nature reviews. Cancer (2018), 18(2), 117-127) in the bulk of the APL tumor cells, it is unclear how these mechanisms contribute to elimination of MRD, particularly given the hypothesized lack of ATRA in the bone marrow niche. It was previously reported that combination of ATRA with ATO may be associated with high incidence of transaminitis and headaches (NEJM (2013), 369(2), 111-121). Since these may be clinical signs of ATRA toxicity, we investigated if addition of ATO changes the pharmacokinetics (PKs) of ATRA in patients with APL. We determined the area under the plasma concentration versus time curve (AUC) for ATRA in patients suspected of APL and treated with single agent ATRA (“preATO”) and in the same patients after the diagnoses of APL was confirmed and the patients received ATO (“postATO”). Even though ATRA should have an expected drop in AUC during the first 3-4 days of therapy (JPET (2017), 361(2), 246-258), addition of ATO resulted in improved ATRA PKs (Figure A). Since ATRA levels are mainly controlled by clearance via hepatic CYP26s, we tested if treatment with ATO changes the levels of CYP26 or its induction by ATRA. We found that exposure to ATO decreases both baseline and ATRA-induced upregulation of CYP26B1 in HepG2 cells (Figure B). Similarly, ATO decreases CYP26B1 levels in human bone marrow derived mesenchymal stroma cells (MSCs) at baseline (21%±7% of NO ATO control, n=3, p<0.01) and after ATRA-induced upregulation (66%±14% of ATRA only control, n=3, p<0.01). These changes resulted in improved ATRA PKs in vitro in the presence of bone marrow MSCs (Figure C). Next, we used NB4-EvAsR cells (NB4R), ATO-resistant APL cells to test if ATO-induced improvement of MSC-dependent ATRA PKs may contribute to elimination of APL MRD. We found that ATO sensitizes NB4R to ATRA in the presence of BM MSCs but not in stroma free conditions (Figure D). ATO had no effect on ATRA-induced differentiation of NB4R in the presence of CYP26B1 knockout stroma (this stroma offers no protection from ATRA) or if stroma expresses CYP26B1 under the control of a viral promoter and thus, resistant to ATO downregulation. This data suggest that ATO-induced changes of BM MSC expression of CYP26 may contribute to elimination of MRD in APL. Stromal CYP26 protects not only APL but also non-APL AML cells from retinoids (Leukemia (2020), 34(11), 3077-3081). Retinoids are synergistic with FLT3 inhibitors to eliminate mutant FLT3 AML cells (Blood (2016), 127(23), 2867-2878) and stromal CYP26 breaks this synergism (Blood (2015) 126 (23): 790). We tested if ATO sensitizes mutant FLT3 AML cells, MV4;11 to gilteritinib in the presence of BM MSCs. For this, we derived MV4.11 cells resistant to ATO (MV4.11R) via long term exposure to increasing concentration of ATO. As seen with NB4R, ATO treatment synergizes with gilteritinib to decrease the IC50 of gilteritinib against the clonogenic activity of MV4.11R in the presence of stroma but not in stroma free conditions (Figure E). In an effort to fully explore the translational potential of these findings, we are currently investigating the mechanism by which ATO changes CYP26 levels.Taken together these findings support a model in which ATO changes the bone marrow niche and it's ability to metabolize retinoids and thus, sensitizes MRD in AML to targeted therapy. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding.

Gilteritinib (GILT) Monotherapy with Addition of Decitabine (DEC) in Non-Responders in Older Newly Diagnosed (ND) FLT3 Mutated Acute Myeloid Leukemia (AML) Patients Having High and Low Variant Allele Frequency (VAF): A Phase 2/1b Sub-Study of the Beat AML Master Trial

Gilteritinib (GILT) Monotherapy with Addition of Decitabine (DEC) in Non-Responders in Older Newly Diagnosed (ND) FLT3 Mutated Acute Myeloid Leukemia (AML) Patients Having High and Low Variant Allele Frequency (VAF): A Phase 2/1b Sub-Study of the Beat AML Master Trial

The Structural Effect of FLT3 Mutations at 835th Position and Their Interaction with Acute Myeloid Leukemia Inhibitors: In Silico Approach.

FMS-like tyrosine kinase 3 (FLT3) gene mutations have been found in more than one-third of Acute Myeloid Leukemia (AML) cases. The most common point mutation in FLT3 occurs at the 835th residue (D835A/E/F/G/H/I/N/V/Y), in the activation loop region. The D835 residue is critical in maintaining FLT3 inactive conformation; these mutations might influence the interaction with clinically approved AML inhibitors used to treat the AML. The molecular mechanism of each of these mutations and their interactions with AML inhibitors at the atomic level is still unknown. In this manuscript, we have investigated the structural consequence of native and mutant FLT-3 proteins and their molecular mechanisms at the atomic level, using molecular dynamics simulations (MDS). In addition, we use the molecular docking method to investigate the binding pattern between the FLT-3 protein and AML inhibitors upon mutations. This study apparently elucidates that, due to mutations in the D835, the FLT-3 structure loses its conformation and becomes more flexible compared to the native FLT3 protein. These structural changes are suggested to contribute to the relapse and resistance responses to AML inhibitors. Identifying the effects of FLT3 at the molecular level will aid in developing a personalized therapeutic strategy for treating patients with FLT-3-associated AML.

An Analysis of Maintenance Therapy and Post-Midostaurin Outcomes in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603/RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations

An Analysis of Maintenance Therapy and Post-Midostaurin Outcomes in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603/RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations

The Addition of Midostaurin to Standard Chemotherapy Decreases Cumulative Incidence of Relapse (CIR) in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603 / RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations

The Addition of Midostaurin to Standard Chemotherapy Decreases Cumulative Incidence of Relapse (CIR) in the International Prospective Randomized, Placebo-Controlled, Double-Blind Trial (CALGB 10603 / RATIFY [Alliance]) for Newly Diagnosed Acute Myeloid Leukemia (AML) Patients with FLT3 Mutations

Comparison of the Profile of the AML Drug Midostaurin (Rydapt®) As a Kinase Inhibitor with Those of Its Predominant Primary Human Metabolites

Background: The multi-targeted kinase inhibitor midostaurin (M) has single agent activity in internal tandem duplication and tyrosine kinase domain mutant FLT3 acute myeloid leukemia (AML). When added to standard chemotherapy M (50 mg, BID) significantly improved both event-free and overall survival in AML patients harboring activating FLT3 mutations (Stone RM, NEJM 2017). In humans, M is converted into 3 primary metabolites: the 10-O-demethylated compound CGP62221 and two epimeric C3-hydroxylated compounds, e1 and e2 (Fig 1). Following the administration of a single 50 mg dose of [14C]-M, the plasma AUC0-96hr of M, CGP62221, e1 and e2 were 18.0, 22.5, 4.12 and 25.8 µg Eq*h/mL respectively (He H, Drug Metab Dispos 2017) and following 50 mg BID the steady-state plasma trough levels of M, CGP62221 and e2 were 0.82, 1.48 and 6.73 µM (Wang Y, J Clin Pharmacol 2008), indicating substantial accumulation of the latter metabolite. Here we compare the kinase profile of M with those of its predominant metabolites.Methods: Reaction of M with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone afforded a mixture of oxidation products. After purification by chromatography (silica gel) and recrysallisation, X-ray crystallography of the more lipophilic epimer (VE12.094; m.p. 298-301°C) confirmed it to have (S)-stereochemistry (Fig 1). Comparison of this epimer with the products from cryopreserved human hepatocyte (BioreclamationIVT, Baltimore, MD, USA)-mediated metabolism of M showed it to be identical (ultra high performance liquid chromatography - mass spectrometry) to e1; the corresponding 3-(R)-epimer (e2) could not be purified. CGP58546 (Hoehn P, J Antibiot 1995) was treated with benzoic anhydride to give CGP62221 (purified by chromatography; m.p. 246°C). The profiles of these compounds as kinase inhibitors were determined using recombinant enzymes (13 lipid and 320 protein kinases) in radiometric transphosphorylation assays (ProQinase GmbH, Freiburg, Germany) and where >50% inhibition was observed at 10 µM, concentrations required to inhibit activity by 50% (IC50 value) were calculated from the dose-response curves.Results: All four compounds showed inhibition of both serine-threonine and tyrosine protein kinases, consistent with their binding in a type-1 ATP-competitive binding mode. M and CGP62221 showed both a similar pattern of selectivity against 320 protein kinases, as well as similar degrees of potency, although CGP62221 (the most potent inhibitor of PRKG2) was less active against LCK (Tables 1 and 2). The presence of a hydroxyl-group in the pyrrole-ring (e1 and e2) generally reduced potency resulting in increased selectivity towards VEGFR2 (none of the compounds evaluated inhibited VEGFR1 by >50% at 10 µM). PDK1 was the only kinase substantially more sensitive to e2 compared with e1 (>3-fold). None of the compounds substantially (>50%) inhibited the activity of any lipid kinase, nor of AKT, nor of any cyclin-dependent protein kinase at 10 µM.Conclusion: The disparate effects of FLT3-targeting drugs in AML clinical trials has been commented upon (Stone RM, Blood 2017). However, the efficacy and side-effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent, but often comprise of complex cooperative effects between the properties of the parent and active metabolites. AML is a heterogeneous malignancy and activating mutations in the FLT3 and KIT receptor kinases are particularly implicated among the mutations driving the malignant phenotype (Patel JP, NEJM 2012). In the case of M, the major circulating metabolites are multi-targeted protein kinase inhibitors with activity against mutant forms of FLT3 and KIT, as well as several kinases involved in their signaling pathways or implicated in AML via stromal support, such as IGF1R (Chapuis N, Haematologica 2010), JAK (Furqan M, Biomarker Res 2013), LYN (Robinson LJ, Exp Hematol 2005), PDK1 (Zabkiewicz J, Haematologica 2014), RET (Rudat S, ASH 2016), SRC (Leischner H, Blood 2012) and SYK (Puissant A, Cancer Cell 2014). Notably, CGP62221, e1 and e2, which are major circulating components following chronic dosing of M, exhibited substantial kinase activity at physiologically relevant doses. A complex interplay between the kinase activities of M and its metabolites may contribute to the efficacy of M in AML and help engender the distinctive effects of M compared to other FLT3 inhibitors in this malignancy. [Display omitted] DisclosuresManley:Novartis International: Employment. Caravatti:Novartis International: Employment. Furet:Novartis International: Employment. Roesel:Novartis International AG: Employment. Tran:Novartis International: Employment. Wagner:Novartis Pharmaceuticals: Employment.

Bone Marrow Stroma Protects FLT3 Acute Myeloid Leukemia (AML) through CYP3A4-Mediated Drug Metabolization of FLT3 Tyrosine Kinase Inhibitors (TKIs)

IntroductionFms-like tyrosine kinase 3 internal tandem duplications (FLT3/ITD) are among the most common mutations found in AML patients. Interestingly, although initial remission rates of FLT3/ITD AML are similar to standard-risk AMLs, these AML have a high relapse rate and poor overall survival. Moreover, FLT3 TKIs have been much more successful in eliminating peripheral FLT/ITD blasts, than eradicating marrow disease and generating durable responses clinically. Studies have addressed several mechanisms underlying the FLT3 TKI resistance, including a protective role for the bone marrow (BM) microenvironment. However, the mechanisms underlying this protection remain unknown. We previously showed that human bone marrow stromal cells (BMSCs) highly express most cytochrome P450 (CYP) enzymes, and these enzymes are a major means of chemoprotection within the BM niche (Alonso et al. Oncotarget 2015). Among them, CYP3A4 is a key enzyme responsible for hepatic inactivation of many chemotherapy agents, including many FLT3 TKIs used clinically. Therefore, we hypothesized that BM stromal CYP3A4 contributes to BM microenvironment mediated FLT3 TKI resistance by FLT3/ITD AML.Methods and ResultsTo test our hypothesis in vitro, FLT3/ITD AML cell lines (MV4-11 and Molm14) were co-cultured with FLT3 TKIs with or without BMSCs (F/STRO), and their clonogenic activity was assessed. We found that 3 different clinically active FLT3 TKIs (sorafenib, quizartinib, and gilteritinib), while active against the FLT3/ITD AML in liquid culture, had little activity when co-cultured with BMSCs (Figure 1A). Moreover, when FLT3/ITD AMLs were co-cultured with BMSCs with short hairpin (sh) RNA knockdown of CYP3A4 (shCYP3A4), the activity of the FLT3 TKIs was restored (Figure 1A). To test the hypothesis in vivo, we investigated the growth of FLT3/ITD AML tumors containing control or shCYP3A4 human primary BMSCs upon FLT3 TKI treatments in a xenograft mouse model. We observed that the tumors with shCYP3A4 BMSCs were significantly more sensitive to FLT3 TKI treatments compared to those with control BMSCs.To further examine the role of CYP3A4 in BMSCs, we examined the inhibitory effects of FLT3 TKIs in the absence of stromal cellular contact with FLT3/ITD AML cells. FLT3 TKI-containing media was incubated with and without control BMSCs and shCYP3A4 BMSCs, and then incubated with FLT3/ITD AML cells. Western blot performed on the FLT3/ITD AML cells showed that FLT3 TKI media conditioned with BMSCs blocked the inhibition of FLT3 phosphorylation, and this was reversed by CYP3A4 knockdown in the BMSCs. These results demonstrated that stromal CYP3A4 protected against FLT3 TKIs even in the absence of stromal cell contact.We next studied if combining a clinically active CYP3A4 inhibitor with FLT3 TKIs would increase the FLT3/ITD AML cells' sensitivities to FLT3 TKIs in the presence of BMSCs. Clarithromycin, a clinically active CYP3A4 inhibitor, significantly reversed the protective effects of BMSCs to 3 different FLT3 TKIs (Figure 1B).ConclusionsThese data confirm the protective effects of stroma for FLT3/ITD AML against FLT3 TKIs. Further, we found for the first time that CYP3A4 expressed in BMSCs contributes to BMSC mediated FLT3/ITD AML protection against FLT3 TKIs both in vitro and in vivo mouse model. Our results also demonstrated that combining FLT3 TKIs with clinically available CYP3A4 inhibitors may become a promising strategy to overcome drug resistance in FLT3/ITD AML treatment. [Display omitted] DisclosuresLevis:Novartis: Consultancy, Honoraria, Research Funding; Daiichi Sankyo, Inc.: Consultancy, Honoraria; Astellas Pharma Us: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; FujiFilm: Research Funding.