FLT3 Internal Tandem Duplication Mutations Research Articles

Real-World Data for FLT3-Mutated Acute Myeloid Leukemia Patients Treated in Resource-Constrained Settings

Background: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a well-established prognostic marker in acute myeloid leukemia (AML), whereas the impact of mutations in the Tirosine-Kinase Domain (TKD) is controversial. The association of TK inhibitors (TKIs) with intensive chemotherapy improved the overall survival (OS) of FLT3-mutated AML patients in randomized clinical trials and is recommended by the European LeukemiaNet panel of experts. Nevertheless, TKIs are not available for most patients treated in Low- and Middle-Income Countries (LMIC) and there is a scarcity of data concerning the outcome of treatment in these resource-constrained settings nowadays. Methods: This is a real-world study encompassing newly diagnosed adults with FLT3-mutated AML intensively treated at public hospitals in Brazil between January 2015 and December 2020, who were registered in the International Consortium on Acute Leukemias (ICAL) ICAML2015 Study. No patient received TKIs as part of the treatment. FLT3 mutation was tested in diagnostic samples by fragment analysis method. We used the Adapted Genetic Risk (AGR) score reported by Silveira et al. (2020), which is an adaptation of the European Leukemia Net (ELN) 2017 classification. The primary endpoints were event-free survival (EFS) and OS, calculated by Kaplan-Meier method. Relapse rate was calculated through competing risk analysis. Results: Overall, 350 pts (median age: 51y; 52.8% females) were included. FLT3-ITD and -TKD mutations were detected in 17.5% and 7.2% of the patients, respectively. Ninety patients (35.1%) were classified in the favorable risk group, 110 (42.9%) in the intermediate, and 56 (21.9%) in the adverse risk group, according to the AGR. The most frequent co-mutation was in the NPM1 gene. The remaining features are summarized in Table 1. Seventy-eight patients underwent allogeneic stem-cell transplantation (HSCT) in first complete remission (22.2%), of whom 27 had FLT3-ITD (i.e. 43.9% of patients harboring FLT3-ITD underwent HSCT). With a median follow-up period of 3.5 years, the median 4-year OS rate was 24.2% (95% CI 19.8-30.1), and the 4-y EFS 22.9% (95% CI 18.6-28.2). When only patients with FLT3-ITD mutations were analyzed, the 4-y OS rate was 14.4% which was inferior to that of patients with wild-type FLT3 (27.5%; p=0.057) (Figure 1). The cumulative incidence of relapse at four years was 39.4% for patients with FLT3-ITD and 43.9% for those with wildtype FLT3 (p=0.5), while the non-relapse mortality (NRM) at four years was 28.1% and 32.4%, respectively. The four-year OS rate for patients with FLT3-ITD mutations who underwent HSCT was 49.1% (95% CI 38.9-62.1). Patients with FLT3-ITD were further subdivided according to the allelic ratio (AR) in those with low (<0.5) and high (³ 0.5), and no significant difference in OS was detected between the two groups. Of note that patients harboring FLT3-TKD mutations showed a better overall survival rate compared to those with wild-type FLT3 (HR 0.49, 95% CI 0.26-0.93, p=0.029), but this result needs to be interpreted cautiously due the small size of the cohort. Conclusion: our results show that the frequency of FLT3-ITD mutations and their association with worse prognosis was similar to those described in high-income countries previously to the introduction of TKI as the standard of care. Therefore, allogeneic HSCT remains a valid consolidation strategy for FLT3-mutated patients. However, less than half of the patients for whom HSCT was indicated were actually transplanted. Additionally, the allelic ratio of FLT3-ITD should no longer be considered a prognostic marker for intensively treated patients not receiving FLT3 inhibitors.

Idarubicin Versus Daunorubicin Versus Mitoxantrone for Induction Chemotherapy in Acute Myeloid Leukemia: Patient Registration Study of Turkish Society of Hematology-Acute Myeloid Leukemia Working Group

Introduction: Standard induction chemotherapy in newly diagnosed acute myeloid leukemia (AML) includes a combination of standard-dose cytarabine and an anthracycline (idarubicin [IDA], daunorubicin [DNR], or mitoxantrone [MXR]). Anthracycline selection is still not standardized, and the impact of anthracycline selection on survival and response in patients with adverse genetic risk is of interest. The study aimed to retrospectively compare the effectiveness of induction regimens with respect to anthracycline preference. Methods: The study population was recruited from the Turkish AML Registry project database based on documentation of newly diagnosed AML and induction chemotherapy with IDA, DAU, or MXR in combination with cytarabine. Patients diagnosed before 31 August 2022 were received typical “7 + 3” induction chemotherapy containing 3 days of IDA, DAU, or MXR. Results: The study included 317 newly diagnosed AML patients (median age 51; range, 18 - 79; 55.2% male). Of the patients, IDA was administered in 266 (83.9%), DNR in 42 (13.2%), and MXR in 9 (2.8%). The three anthracycline treatment groups were similar between age, gender, type of AML by the World Health Organization (WHO), and risk classification by 2017 European LeukemiaNet (ELN) when evaluated according to the baseline disease characteristics. At the time of diagnosis, although the median white blood cell levels were 19 x 10 9/L (0.4 - 355) in the IDA, 7.8 x 10 9/L (0.7 - 357.2) in the DAU, and 5.5 x 10 9/L (0.6 - 34) in the MXR therapy group (p = 0.019); the hemoglobin and platelet levels and bone marrow blast percentage were similar between the treatment groups. FMS-like tyrosine kinase-3 internal tandem duplication ( FLT3-ITD) (p = 0.561) and Nucleophosmin 1 gene ( NPM1) (p = 0.430) mutation frequencies were also similar between treatment groups. Morphological complete remission (CR) was documented in 65% of all evaluable patients. Among the patient groups, CR was 67.7% for IDA, 50% for DNR, and 55.6% for MXR (p = 0.081). In univariate analysis (Table 1), significant predictors of overall survival (OS) were age ≥60 years (p <0.001), acute leukemias of ambiguous lineage (p <0.001), presence of disseminated intravascular coagulation (DIC) (p = 0.012), and FLT3-ITD mutation (p = 0.018); no significant interaction was noted between NPM1 mutation and OS (p = 0.059). Additionally, when idarubicin was taken as a reference, there was no meaningful relationship between anthracycline type and OS (p values for daunorubicin and mitoxantrone were 0.145 and 0.356, respectively). In multivariate analysis (Table 1), age ≥60 years (p = 0.001), acute leukemias of ambiguous lineage (p = 0.007), and the presence of DIC (p = 0.040) remained significant. After a median follow-up of 29.8 months, 32.5% relapse cases were documented and similarly distributed among IDA (33.1%), DNR (31%), and MXR (22.2%) therapy groups (p = 0.771). During the same period, allogeneic hematopoietic stem cell transplantation (AHSCT) was documented in 10.9% and 26.2% of patients treated for IDA and DNR, respectively. No patient progressed to AHSCT in the MXR arm (p <0.001). In the study, 142 (44.8%) deaths were reported, and there was a 48.5%, 26.2%, and 22.2% mortality rate between the IDA, DNR, and MXR treatment groups, respectively. There was no significant difference in OS by anthracycline type in both the study cohort and age group <60 years (Figures 1A and 1B). There was also no significant difference in OS in patients with the adverse genetic risk group and <60 years age group with adverse genetic risk, according to 2017ELN (Figures 1C and 1D). The results may have been influenced by the number of patients in the treatment groups of the retrospective study and by differences in fitness levels that needed to be measured systematically. Conclusions: We are encouraged that our findings mostly agree with previously published studies and that IDA, DNR, and MXR offer comparable survival values in the research and the younger patient groups. In the future, the ever-changing treatment environment in patient groups with targetable mutations makes choosing the optimal anthracycline for induction chemotherapy in AML much more difficult.

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

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

Improvement in Survival of Patients with FLT3 Mutated Acute Myeloid Leukemia: Results from a Retrospective Canadian Cohort

Introduction: FLT3 internal tandem duplication (ITD) mutations have been historically associated with inferior outcomes in patients with acute myeloid leukemia (AML) and an intermediate risk status as per the European LeukemiaNet (ELN) 2022 guidelines. In 2017, the US FDA granted approval to midostaurin for frontline treatment and a year later, gilteritinib for use in relapsed/refractory (R/R) AML. These agents began to be widely used in our provincial leukemia program in early 2018 as per the label indications. In this study, we compared clinical outcomes for patients with FLT3-ITD mutated AML before and after this change in British Columbia. Methods: We identified patients in our program database diagnosed with FLT3-ITD mutated AML from January 1, 2010 to December 31, 2021. Only patients receiving intensive chemotherapy with cytarabine and anthracycline were included. Patients were divided into two eras: 2010-2017 (era 1) and 2018-2021 (era 2). The two eras were compared in terms of patient characteristics, genetics, rate of complete remission (CR) and CR with incomplete count recovery (CRi), receipt of allogeneic stem cell transplant (alloSCT) and the overall and relapse free survival (OS and RFS). OS was calculated from diagnosis to death from any cause and was not censored at the time of alloSCT. RFS was calculated from CR/CRi to relapse. Patients without an event during the study period were censored at the time of last follow-up. Categorical variables were compared using Chi-square and Fisher exact tests and continuous variable were compared using paired T tests. Kaplan-Meier survival method and log rank test were used to estimate and compare survival. Uni- and multivariate analyses were performed in a Cox proportional regression model and alloSCT was considered as a time dependent covariate. Results: 169 patients were included (era 1=101, era 2=68). The median duration of follow up of the entire cohort was 17 months (range 0.5-148.6 months) and follow-up of surviving patients (n=70) was longer in era 1 vs. era 2 (median 88 months vs. 32 months, p<0.001). The median age of the entire cohort was 60 years (range 26-79 years) and it was comparable in both eras. Most patients had normal cytogenetics (era 1: 76%; era 2: 69%). NPM1 was mutated in 69/101 (68%) and 38/68 (56%) patients in eras 1 and 2 respectively. Due to lack of genetic data for era 1, we did not risk stratify based on the recent ELN classification. The rate of CR/CRi was 58/101 (57%) vs. 40/68 (59%) after induction chemotherapy and 78/101 (77%) vs. 58/67 (87%) (p=0.09) at any time in eras 1 and 2 respectively. AlloSCT was performed in 58% and 72% of patients in the two cohorts. Midostaurin was used in 82% and gilteritinib was used in 26% of era 2 patients in the second era. OS was significantly longer in era 2 (median OS 73 months vs. 11 months, HR (death) 6.2, p=0.012) (Figure). The 2-year OS rate was higher in era 2 (57%) than in era 1 (39%). In patients achieving CR/CRi, median RFS was longer in era 2 (51 vs. 16 months, p=0.43), although this was not statistically significant. In univariate Cox regression analysis the following factors were associated with improved OS: use of midostaurin (HR 0.487, 95% CI 0.304-0.779, p=0.003), diagnosis in era 2 (HR 0.583, 95% CI 0.38-0.895, p=0.014) and receipt of alloSCT (HR 0.346,) 95% CI 0.219-0.545, p<0.001). Other parameters including age, sex, NPM1 status and cytogenetics were not significant. In multivariate analysis including era, midostaurin and alloSCT, only the receipt of alloSCT was associated with significantly improved OS (HR 0.362, 95% CI 0.229 - 0.572, p<0.001) whereas use of midostaurin (HR=0.465, CI 0.207-1.042, p=0.063) and era (HR 1.12, 95% CI 0.538-2.34, p=0.76) were not significant. Conclusions: Our results show thatOS for patients with FLT3-ITD AML has improved in patients diagnosed in a more recent treatment era. Patients diagnosed after 2018 were more frequently treated with AlloSCT and FLT3-inhibitors, which may have resulted in improvements in OS. Our findings support the assignment of FLT3-ITD AML to theintermediate risk category as per the current ELN guidelines. A limitation of this study is the differences in follow-up time between eras, which may influence the event rate between the groups.

Persistence of FLT3-TKD in Blood Prior to Allogeneic Transplant Is Associated with Increased Relapse and Death in Adults with AML in First Remission

Introduction: Next-generation sequencing (NGS) represents a promising modality for measurable residual disease (MRD) testing in patients with acute myeloid leukemia (AML), but systematic efforts are required to define the appropriate targets, test characteristics, and clinical implications. We recently reported that detection of persistent NPM1 or FLT3 internal tandem duplication (ITD) mutations in adults with AML in first complete remission (CR1) prior to allogeneic hematopoietic cell transplantation (alloHCT) is associated with increased relapse and death compared with those testing negative (JAMA 2023, PMID: 36881031). FLT3 tyrosine kinase domain (TKD) variants are present in 7-10% of adult patients with AML but the utility of this target for AML MRD testing in CR1 prior to alloHCT was previously unknown. Methods: Patients aged 18 or older who underwent alloHCT for AML in CR1 between 2013 and 2019 reported to have a FLT3-TKD variant at diagnosis and a pre-conditioning remission blood sample available in the CIBMTR biorepository were eligible for this study. Single-amplicon NGS (SA-NGS) library generation utilizing UMI-tagged primers targeting the D835 and I836 codons of FLT3 was performed on 400ng genomic DNA sequenced with unique dual indices, with error-corrected variant calling. Primary outcomes were overall survival (OS) and cumulative incidence of relapse (CIR). Secondary outcomes were relapse-free survival (RFS) and non-relapse mortality (NRM), with NRM being considered as a competing risk. Results: A total of351 patients met inclusion criteria for this study, and 342 (97.4%) had sufficient DNA for SA-NGS and were analyzed for clinical outcomes. Of these patients, 81 (23.7%) experienced relapse at a median of 5 months post-alloHCT. Using the current ELN-recommended variant allele fraction (VAF) threshold of 0.1% for NGS-based AML MRD, 14 patients (4.1%) tested positive. Patients with FLT3-TKD MRD VAF≥0.1% experienced higher rates of relapse (73.8% vs. 20.5%, p < 0.0001) and decreased OS (11.4% vs. 66.8%, p < 0.001) compared to VAF<0.1%. The highly sensitive SA-NGS method allowed for exploration to determine if a deeper VAF threshold of 0.01% (previously validated for NPM1 and FLT3-ITD in the Pre-MEASURE study) was prognostic for FLT3-TKD as an MRD target. This led to an additional 20 patients being reclassified as positive (n = 34 with VAF above 0.01%, =9.9%). However, there was no significant difference in CIR (33.6% vs. 19.7%, p = 0.089) or OS (65.2% vs. 66.9%, p = 0.267) between patients with 0.01%≤VAF<0.1% and VAF<0.01% ( Figure). Out of 28 SA-NGS positive calls, 6 could not be validated by orthogonal digital droplet PCR testing, and interestingly, none of those 6 patients experienced relapse. Site-reported pre-transplant remission multiparametric flow cytometry (MFC) was available for 335 patients (98%). Results of MFC were not associated with differences in CIR (HR 1.2, 95% CI 0.52 - 2.76, p = 0.67) or OS (HR 0.78, 95% CI 0.34 - 1.77, p = 0.551).In multivariable analysis, FLT3-TKD NGS-MRD VAF≥0.1% remained prognostic for CIR (HR 6.1, 95% CI 3.1 - 12.0, p < 0.001) and OS (HR 3.2, 95% CI 1.7 - 5.9, p < 0.001). Conclusions: Detectable persistence of FLT3-TKD variants in blood from AML patients in first remission prior to first alloHCT is rare, but at a VAF level of 0.1% was strongly associated with increased relapse and death after transplant compared to those testing negative. In contrast to FLT3-ITD NGS-MRD, no evidence was found to support lowering the NGS-MRD VAF threshold from 0.1% to 0.01% for FLT3-TKD. Alternative methodology may further improve NGS-MRD test performance. Site-reported MFC prior to alloHCT did not stratify for post-transplant relapse or survival. Optimal NGS-MRD testing for patients with AML will likely require a multi-target approach and testing for persistence of FLT3-TKD MRD is shown here to represent an important component of this strategy.

Children's Oncology Group's 2023 blueprint for research: Myeloid neoplasms.

During the past decade, the outcomes of pediatric patients with acute myeloid leukemia (AML) have plateaued with 5-year event-free survival (EFS) and overall survival (OS) of approximately 46 and 64%, respectively. Outcomes are particularly poor for those children with high-risk disease, who have 5-year OS of 46%. Substantial survival improvements have been observed for a subset of patients treated with targeted therapies. Specifically, children with KMT2A-rearranged AML and/or FLT3 internal tandem duplication (FLT3-ITD) mutations benefitted from the addition of gemtuzumab ozogamicin, an anti-CD33 antibody-drug conjugate, in the AAML0531 clinical trial (NCT00372593). Sorafenib also improved response and survival in children with FLT3-ITD AML in the AAML1031 clinical trial (NCT01371981). Advances in characterization of prognostic cytomolecular events have helped to identify patients at highest risk of relapse and facilitated allocation to consolidative hematopoietic stem cell transplant (HSCT) in first remission. Some patients clearly have improved survival with HSCT, although the benefit is largely unknown for most patients. Finally, data-driven refinements in supportive care recommendations continue to evolve with meaningful and measurable reductions in toxicity and improvements in EFS and OS. As advances in application of targeted therapies, risk stratification, and improved supportive care measures are incorporated into current trials and become standard-of-care, there is every expectation that we will see improved survival with a reduction in toxic morbidity and mortality. The research agenda of the Children's Oncology Group's Myeloid Diseases Committee continues to build upon experience and outcomes with an overarching goal of curing more children with AML.

Rational design of 4-((6-phenoxypyrimidin-4-yl)amino)-N-(4-(piperazin-1-yl)phenyl)-1H-pyrazole-3-carboxamide (LT-540-717) as orally bioavailable FLT3 inhibitor

In recent years, fms-like tyrosine kinase 3 (FLT3) was confirmed as an exciting target for treatment of AML. However, resistance to FLT3 inhibitors caused by acquired point mutations in tyrosine kinase domain (TKD) have limited their sustained efficacious. Thus, there remains an unmet need to develop high-efficacy FLT3 inhibitors against both FLT3 internal tandem duplication (ITD) and FLT3 (TKD) mutations. Herein, we describe the discovery of compound LT-540-717 (32), a potent FLT3 inhibitor (IC50: 0.62 nM), starting from FN-1501. Compound 32 exhibited highly inhibitory activity against several acquired FLT3 mutations including FLT3 (ITD, D835V), FLT3 (ITD, F691L), FLT3 (D835Y) and FLT3 (D835V). Additionally, 32 displayed potent antiproliferative activity against FLT3-mutation driven BaF3 and AML cells. Oral administration of 32 (25 mg/kg, QD) significantly prohibited tumor growth (tumor-inhibition rate is 94.18%), and no obvious side effect was observed even when increasing dose to 50 mg/kg (tumor-inhibition rate is 93.98%). Furthermore, 32 showed an acceptable bioavailability (F = 33.3% in rat and 72.7% in beagles), a suitable half-life time (T1/2 = 3.5 h in rat and T1/2 = 11.1 h in beagles), and a satisfactory metabolic stability. In summary, these results show the therapeutic potential of 32 to become a new anti-AML drug, especially for AML harboring dual FLT3 (ITD, TKD) mutations.

Oncogenic FLT3 internal tandem duplication activates E2F1 to regulate purine metabolism in acute myeloid leukaemia

Oncogene FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for 30 % of acute myeloid leukaemia (AML) cases and induces transformation. Previously, we found that E2F transcription factor 1 (E2F1) was involved in AML cell differentiation. Here, we reported that E2F1 expression was aberrantly upregulated in AML patients, especially in AML patients carrying FLT3-ITD. E2F1 knockdown inhibited cell proliferation and increased cell sensitivity to chemotherapy in cultured FLT3-ITD-positive AML cells. E2F1-depleted FLT3-ITD+ AML cells lost their malignancy as shown by the reduced leukaemia burden and prolonged survival in NOD-PrkdcscidIl2rgem1/Smoc mice receiving xenografts. Additionally, FLT3-ITD-driven transformation of human CD34+ hematopoietic stem and progenitor cells was counteracted by E2F1 knockdown. Mechanistically, FLT3-ITD enhanced the expression and nuclear accumulation of E2F1 in AML cells. Further study using chromatin immunoprecipitation-sequencing and metabolomics analyses revealed that ectopic FLT3-ITD promoted the recruitment of E2F1 on genes encoding key enzymatic regulators of purine metabolism and thus supported AML cell proliferation. Together, this study demonstrates that E2F1-activated purine metabolism is a critical downstream process of FLT3-ITD in AML and a potential target for FLT3-ITD+ AML patients.

Comprehensive molecular understanding of pediatric acute myeloid leukemia

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease with various genetic abnormalities. Recent advances in genetic analysis have enabled the identification of causative genes in > 90% of pediatric AML cases. Fusion genes such as RUNX1::RUNX1T1, CBFB::MYH11, and KMT2A::MLLT3 are frequently detected in > 70% of pediatric AML cases, whereas FLT3-internal tandem duplication, CEBPA-bZip, and NPM1 mutations are detected in approximately 5-15% of cases, respectively. Conversely, mutations in DNMT3A, TET2, and IDH, which are common in adults, are extremely rare in pediatric AML. The genetic characteristics of pediatric AML are slightly different from those of adult AML. For accurate risk stratification and treatment intensity, genome analysis should be performed in a simple, fast, and inexpensive manner and the results should be returned to patients in real time. As with acute lymphoblastic leukemia, the presence or absence of minimal residual disease is an important factor in determining the success of treatment against AML, and it is important to predict prognosis and formulate treatment strategies considering the genetic abnormalities. For the development and clinical application of new molecularly targeted therapies based on identified genetic abnormalities, it is necessary to explore when and in which combinations drugs will be most effective.

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.

Outcomes in Patients with FLT3-Mutated Relapsed/ Refractory Acute Myelogenous Leukemia Who Underwent Transplantation in the Phase 3 ADMIRAL Trial of Gilteritinib versus Salvage Chemotherapy

The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib improved the survival of patients with relapsed or refractory (R/R) FLT3-mutated acute myelogenous leukemia (AML) in the phase 3 ADMIRAL trial. In this study, we assessed survival and relapse rates of patients in the ADMIRAL trial who underwent hematopoietic stem cell transplantation (HSCT), as well as safety outcomes in patients who received post-transplantation gilteritinib maintenance therapy. ADMIRAL was a global phase 3 randomized controlled trial that enrolled adult patients with FLT3-mutated R/R AML. Patients with R/R AML who harbored FLT3 internal tandem duplication mutations in the juxtamembrane domain or D835/I836 point mutations in the tyrosine kinase domain were randomized (2:1) to gilteritinib (120 mg/day) or to preselected high- or low-intensity salvage chemotherapy (1 or 2 cycles). Patients in the gilteritinib arm who proceeded to HSCT could receive post-transplantation gilteritinib maintenance therapy if they were within 30 to 90 days post-transplantation and had achieved composite complete remission (CRc) with successful engraftment and no post-transplantation complications. Adverse events (AEs) during HSCT were recorded in the gilteritinib arm only. Survival outcomes and the cumulative incidence of relapse were assessed in patients who underwent HSCT during the trial. Treatment-emergent AEs were evaluated in patients who restarted gilteritinib as post-transplantation maintenance therapy. Patients in the gilteritinib arm underwent HSCT more frequently than those in the chemotherapy arm (26% [n = 64] versus 15% [n = 19]). For all transplantation recipients, 12- and 24-month overall survival (OS) rates were 68% and 47%, respectively. Despite a trend toward longer OS after pretransplantation CRc, post-transplantation survival was comparable in the 2 arms. Patients who resumed gilteritinib after HSCT had a low relapse rate after pretransplantation CRc (20%) or CR (0%). The most common AEs observed with post-transplantation gilteritinib therapy were increased alanine aminotransferase level (45%), pyrexia (43%), and diarrhea (40%); grade ≥3 AEs were related primarily to myelosuppression. The incidences of grade ≥III acute graft-versus-host disease and related mortality were low. Post-transplantation survival was similar across the 2 study arms in the ADMIRAL trial, but higher remission rates with gilteritinib facilitated receipt of HSCT. Gilteritinib as post-transplantation maintenance therapy had a stable safety and tolerability profile and was associated with low relapse rates. Taken together, these data support a preference for bridging therapy with gilteritinib over chemotherapy in transplantation-eligible patients.

FLT3ITD drives context-specific changes in cell identity and variable interferon dependence during AML initiation

AbstractAcute myeloid leukemia (AML) initiation requires multiple rate-limiting mutations to cooperatively reprogram progenitor cell identity. For example, FLT3 internal tandem duplication (FLT3ITD) mutations cooperate with a variety of different initiating mutations to reprogram myeloid progenitor fate. These initiating mutations often skew toward either pediatric or adult AML patient populations, though FLT3ITD itself occurs at similar frequencies in both age groups. This raises the question of whether FLT3ITD might induce distinct transcriptional programs and unmask distinct therapeutic vulnerabilities when paired with pediatric, as opposed to adult AML-initiating mutations. To explore this possibility, we compared AML evolution in mice that carried Flt3ITD/NUP98-HOXD13 (NHD13) or Flt3ITD/Runx1DEL mutation pairs, which are respectively most common in pediatric and adult AML. Single-cell analyses and epigenome profiling revealed distinct interactions between Flt3ITD and its cooperating mutations. Whereas Flt3ITD and Flt3ITD/Runx1DEL caused aberrant expansion of myeloid progenitors, Flt3ITD/NHD13 drove the emergence of a pre-AML population that did not resemble normal hematopoietic progenitors. Differences between Flt3ITD/Runx1DEL and Flt3ITD/NHD13 cooperative target gene expression extended to fully transformed AML as well. Flt3ITD/NHD13 cooperative target genes were enriched in human NUP98-translocated AML. Flt3ITD/NHD13 selectively hijacked type I interferon signaling to drive expansion of the pre-AML population. Blocking interferon signaling delayed AML initiation and extended survival. Thus, common AML driver mutations, such as FLT3ITD, can coopt different mechanisms of transformation in different genetic contexts. Furthermore, pediatric-biased NUP98 fusions convey actionable interferon dependence.

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

Prognostic Impact of NPM1 and FLT3-ITD Mutations in Patients Treated with Non-Intensive Regimens: A Pethema Registry Study

Introduction: Acute myeloid leukemia (AML) is the most common acute leukemia of adults, with median age at diagnosis of 68 years. Mutations in FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) are associated with a higher risk of relapse and inferior overall survival (OS) in younger patients treated with intensive chemotherapy, but the prognostic relevance among older patients treated with non-intensive approaches is not well established. As well, the role of nucleophosmin (NPM1), and its interaction with FLT3-ITD status and allelic ratio (AR) remains unclear among elderly. We aimed to assess the prognostic impact of FLT3-ITD and NPM1 mutations in older/unfit newly diagnosed AML patients treated with non-intensive front-line therapies. Methods: We conducted a retrospective analysis that included 707 patients treated with different non-intensive regimens in the PETHEMA AML registry, between 2007 and 2020. FLT3-ITD and NPM1 mutations were studied by fragment length analysis. The karyotype information was available in 615 patients. Associations of demographic and clinical parameters in patients harboring FLT3-ITD and NPM1 mutations was performed using Mann-Whitney-U-test for continuous and Chi-square test for discrete variables. Overall survival (OS) and relapse curves were constructed using Kaplan-Meier and a log-rank test. All analyses were performed by SPSS. Results: The median age was 74 years (range: 43-91), and 410 patients (58%) were male. ECOG status was 0/1 in 474 patients (72%) and 2 in 124 patients (18.9%). FLT3-ITD mutation was identified in 99 patients (14%). When available, 33 patients (40.7%) showed FLT3-ITDlow (AR <0.5). NPM1 mutation was detected in 144 patients (20%). Karyotype was abnormal in 301 patients (42.7%). Regarding the frontline therapy 328 (46.3%) patients received hypomethylating agents (HMA); 353 (49.9%) fludarabine + low dose cytarabine (LDAC) (FLUGA); 11 (1.6%) fludarabine + LDAC + oral idarubicin (FLAG-IDA Lite); and 15 (2.1%) LDAC. Mortality at 2 years was 77.6% and median OS 6 months. FLT3-ITD patients (N=99) showed a non-significant difference in OS compared to FLT3 wild-type (FLT3WT) patients (N=608): median OS 5 vs 7.3 months (p=0.17). There were no differences when comparing low and high AR: FLT3-ITDlow (N=34) vs FLT-ITDhigh (N=47): median OS 5.5 vs 4.9 months (p=0.64). Survival in NPM1-mutated patients (N=144) was similar than in NPM1WT patients (N=519): median OS 7.2 vs 6.8 months (p=0.24). OS according to ELN 2017 risk stratification, considering only NPM1 and FLT3-ITD mutations, was similar in subgroups low risk (N=117), intermediate risk (N=31), high risk (N=23): median OS 7.6 vs 5.4 vs 7.2 (p=0.26) (Fig. 1). In the subgroup of normal karyotype (N=314), FLT3-ITD patients (N=64) showed a trend towards lower survival as compared to FLT3WT patients (N=249): median OS 5.4 vs 10.9 months (p=0.058), with no impact of AR: FLT3-ITDlow (N=19) vs. FLT3-ITDhigh (N=32) median OS 5.8 vs 4.8 months (p=0.73). NPM1 mutated patients (N=102) showed a non-significant difference with NPM1WT patients (N=196): median OS 7.2 vs 10.3 months (p=0.66). OS according to ELN 2017 considering only NPM1 and FLT3-ITD status was similar in subgroups: low risk (N=80), intermediate risk (N=24), high risk (N=12): median OS 2.3 vs 11 vs 2.5 months, respectively (p=0.64). AML patients treated with azacitidine (N=281) had longer median OS of 10 months (p=0.005; 95% CI 1.03 to 1.37) vs decitabine (N=47) median OS 6.1 months and FLUGA median OS 5 months (N=352) (Fig. 2). Conclusions: Data from this large cohort of AML patients treated with non-intensive regimens underlines the limitations of the ELN 2017 risk stratification in this setting. Nonetheless, there was a trend towards worse OS in patients with normal karyotype and FLT3-ITD mutation (irrespectively of the allelic burden) as compared to FLT3WT patients. Figure 1. Overall survival according to ELN 2017 risk stratification Figure 2. Overall survival regarding non-intensive regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Risk Stratification Model for Cytogenetically Normal Acute Myeloid Leukemia with FLT3-ITD Mutation

Introduction Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are observed in about 30 % of patients with newly diagnosed cytogenetically normal acute myeloid leukemia (CN-AML) and has now been acknowledged to be associated with a poor survival. However, long-term follow-up revealed a high degree of heterogeneity in the clinical prognosis of these patients. Further risk stratification to identify high-risk patients is urgently needed. Methods A total of 106 patients with CN-AML and FLT3-ITD mutations who visited the People's Hospital of Peking University from 2007-2019 were included, and target region sequencing was performed by 266 gene panel. Patients were randomly divided into training and validation cohort according to the ratio of 7:3. Molecular and clinical data in training cohort were incorporated to construct a nomogram model. Cumulative incidence of relapse (CIR), Relapse-free survival (RFS) and survival by allogenic hematopoietic stem cell transplantation (allo-HSCT) and consolidation chemotherapy were compared. Results Clinical variables, genes, and genetic groups with mutations occurring in more than 10 patients were enrolled for univariate analysis of RFS (allo-HSCT was recorded as a censored event). And to avoid bias patients with RFS ≤ 4.57 months (median interval from complete remission to allo-HSCT) would be excluded from the subsequent analyses unless receiving an allo-HSCT was treated as a censored event. Three variables were eventually entered into the nomogram analysis: DNMT3A mutation (+ vs. -), minimal residual disease detected by multiparameter flow cytometry (MPFC-MRD) at the end of the 2nd course of consolidation chemotherapy (CON2) (+ vs. -, represents ≥ vs. ＜0.01%), typical ITDs (+ vs. -, represents ITDs have insertion sequences that exactly match wild-type FLT3 gene vs. ITDs have insertion sequences containing other exogenous nucleotides). And the model demonstrated good discrimination in both the training and validation cohort with C-index value of 0.82 (95% confidence interval, 0.7-0.86), 0.86 (95% confidence interval, 0.76-0.95), respectively. The Nomogram model was constructed as: Score = 1.44×DNMT3A mutation+2.16×MFPC-MRD-0.94×typical ITDs (Figure 1, A). According to the variables in nomogram model, patients with risk scores ≤ 0 were assigned to the low-risk subgroup (N = 39) and the rest were assigned to the high-risk subgroup (N = 67). The 5-year RFS was significantly higher in the low-risk group than in the high-risk group (training cohort, HR 3.68 [1.16-11.66], P = 0.002; validation group, HR 4.45 [1.52-13.00], P = 0.03) (Figure 1, B-C). We then interrogated the effect of consolidation chemotherapy and allo-HSCT as post-remission therapies in the two risk subgroups. Allo-HSCT did not further improve the prognosis of patients in the low-risk group (allo-HSCT vs. chemotherapy, 5-year CIR, HR 0.46 [0.11-1.93], P = 0.29; 5-year RFS, HR 0.41 [0.09-1.86], P = 0.24; 5-year survival, HR 0.91 [0.18-4.68], P = 0.91) (Figure 2, A-D). In contrast, in the high-risk group of patients, transplantation showed a significant survival benefit (5-year CIR, HR 0.21 [0.06-0.74], P = 0.01; 5-year RFS, HR 0.23 [0.06-0.80], P = 0.01; 5-year survival, HR 0.25 [0.06-1.11], P = 0.05) (Figure 2, E-H). Conclusion Our study suggests that risk stratification by our novel nomogram scoring system can identify patients at high risk of relapse in CN-AML with FLT3-ITD mutations and allo-HSCT further benefits their prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical and Molecular Response Assessment of AML Harboring Non-Canonical FLT3 p.N676K Driver Mutations to FLT3 Inhibitor Therapies

Treatment of acute myeloid leukemia (AML) has been enhanced by the development and regulatory approval of a series of novel agents, including midostaurin and gilteritinib (FLT3 inhibitors), venetoclax (BCL2 inhibitor), ivosidenib (IDH1 inhibitor), and enasidenib (IDH2 inhibitor). A difficulty that has arisen in the era of molecular therapies, however, is determining the efficacy of these agents for patients with AML harboring atypical driver mutations. These atypical drivers were underrepresented in seminal clinical trials that led to the approval of targeted AML therapies, thereby limiting the availability of data for clinical decision making. For example, the non-canonical FLT3 p.N676K variant was initially described as an acquired resistance mechanism in patients with FLT3 internal tandem duplication (ITD) mutations who were treated with midostaurin. Follow-up studies in Ba/F3 cells demonstrated that the FLT3 N676K mutation is sensitive to midostaurin and quizartinib, but that co-occurrence of FLT3 N676K and ITD mutations confers resistance to both targeted agents. Clinical data from patients with the FLT3 N676K mutation, however, is limited to reports of two patients, both of whom developed the N676K mutation at relapse. There have been no published reports detailing the treatment of patients with the FLT3 N676K mutation with de novo disease, and limited data exist regarding the role of FLT3 inhibition for patients with the FLT3 N676K mutation in the clinical setting. To address this knowledge gap, we detail our experience caring for nine individuals with AML harboring FLT3 N676K at The University of Chicago. The patients were identified by querying the University of Chicago molecular pathology database as part of an IRB-approved retrospective analysis. Six of these patients had AML with FLT3 N676K arising as the only FLT3 variant, whereas the mutational composition of three patients carried coincident ITD or tyrosine kinase domain (TKD) mutations at some point during their clinical course. The median age at diagnosis of AML in our cohort was 41 years, with a mean presenting WBC count of 53.3 k/uL. Four of nine patients had normal cytogenetics. Additional demographic and disease-specific parameters are depicted in Table 1. One elderly patient pursued comfort care immediately after presentation. Of the remaining eight patients, six received FLT3 inhibitors at any point during their clinical trajectory. Three patients were treated with FLT3 inhibitors added to upfront standard induction therapy (midostaurin, n=2 ; sorafenib, n=1). Of those, one (patient #1) achieved a morphologic and molecular complete remission and proceeded to curative-intent allogeneic transplant. The remaining two patients (patients #5 and #6) harbored co-occuring FLT3 mutations, one with FLT3 ITD and the other with FLT3 TKD, and both experienced relapse after an initial complete remission following induction therapy. In patients for whom longitudinal next-generation sequencing data from bone marrow specimens was available, we observed dramatic reduction in measurable FLT3 N676K variant allele frequencies after FLT3 inhibitor use, regardless of line of therapy (Fig. 1). Here, we describe the largest series to date of AML patients with atypical FLT3 N676K driver mutations. Affected individuals in our cohort presented at a notably younger age and demonstrated reductions in disease burden with the use of targeted FLT3 inhibitors. The presence of concurrent canonical FLT3 mutations was associated with loss of treatment response. Longer-term follow will help evaluate the durability of FLT3 inhibitor maintenance strategies in both transplant and non-transplant clinical settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Inhibitor of FLT3 and Its Drug-Resistant Mutants with Superior Activity to Gilteritinib in Molm-13 Preclinical Acute Myeloid Leukemia Xenograft Model

Acute Myeloid Leukemia (AML) is a complex heterogenous disease characterized by the aberrant growth of undifferentiated myeloid progenitor cells typically driven by a combination of epigenetic and signaling mutations. In particular, fms-like tyrosine kinase 3 (Flt3) internal tandem duplication (FLT3-ITD) is associated with poor relapse-free and overall survival. Several FLT3 inhibitors have been developed such as the first generation multikinase inhibitor Midostaurin which is approved for newly diagnosed FLT3 mutant AML in combination with induction chemotherapy. Second generation inhibitors such as gilteritinib are more selective and potent and are also approved for relapsed/refractory FLT3 mutant AML. Although initial response has been positive with this class of agents, prolonged remission is rarely achieved, and diverse mechanisms of resistance often arise with FLT3 inhibitors representing an ongoing challenge. Common mechanisms of resistance to FLT3 inhibitors includes the acquisition of tyrosine kinase domain (TKD) secondary mutations such as the gatekeeper F691L TKD mutation, or the emergence of additional mutations in alternative signaling pathways such as RAS/MAPK. Thus, it is important to continue developing novel inhibitors capable of circumventing these resistance mechanisms. Herein we describe 2082-0047, a novel tyrosine kinase inhibitor with sub-nanomolar potency against FLT3 mutant AML, including TKD mutations. Compound 2082-0047 is a highly selective pan-FLT3 kinase inhibitor, as in both scanMAX and KdELECT KINOMEscan™ Profiling assays (Eurofins DiscoverX) it has shown 400-1000 fold selectivity. In our initial screening, 2082-0047 was only effective in FLT3-ITD mutant cells, however the KINOMEscan™ Profiling data described above demonstrates that 2082-0047 is potent against FLT3, FLT3 ITD, D835H, D835V, D835Y, D835V, K663Q, N841I, and R834Q FLT3 TKD mutations. We confirmed this data in a cellular assay using BaF3 cell lines expressing the TKD mutations D835Y, D835H or F691L alone or with Flt3-ITD. Next, we confirmed using immunoblot analysis of phosphorylated Flt3 that 2082-0047 is a potent inhibitor of FLT3-ITD signaling (superior to gilteritinib) in FLT3-ITD mutant AML cell lines. Using wildtype B6 mice, we conducted pharmacokinetic (PK) studies and found that 2082-0047 had 79 % oral bioavailability, achieved 1.2 μΜ Cmax after 4 hours, and had half-life of 4.7 hours. Next, our absorption, distribution, metabolism and excretion (ADME) studies showed lack of CYP inhibition of major human isoforms. Overall, our PK studies demonstrated overall favorable drug like properties enabling in vivo investigation of therapeutic activity. Therefore, we proceeded with using the MOLM-13 disseminated xenograft model in NCG recipient mice to determine the overall survival of 2082-0047 compared to gilteritinib. We enrolled mice 10 days post engraftment to receive daily oral vehicle, 3, 10, 20 or 30 mg/kg of 2082-0047 or 30 mg/kg of gilteritinib and found that 10 and 20 mg/kg of 2082-0047 were well tolerated and superior to gilteritinib at 30 mg/kg (Figure 1). In summary, we describe a novel and potent inhibitor of FLT3, including both internal tandem duplication and drug-resistant tyrosine kinase domain (TKD) mutants, with promising preclinical activity which is superior to the most potent FDA approved FLT3 inhibitor gilteritinib in an aggressive AML in vivo model. Our non-GLP in vivo safety studies confirmed a broad therapeutic window of 2082-0047. Ongoing Investigational New Drug (IND) enabling studies will include comparing the effect of 2082-0047 to gilteritinib in an immunocompetent syngeneic AML murine model harboring both a Flt3-ITD and F691 TKD gatekeeper mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bmf-500: An Orally Bioavailable Covalent Inhibitor of FLT3 with High Selectivity and Potent Antileukemic Activity in FLT3-Mutated AML

Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) are the most frequent genetic alteration in AML and are associated with poor prognosis. Though several FLT3 inhibitors have entered clinical trials and reached commercialization, adverse events and dose-limiting toxicities often restrict the therapeutic window and limit their long-term use. Such limitations impact the ability to achieve long-lasting response in patients and ultimately result in therapy-induced resistance. Exquisite potency combined with high selectivity and improved safety profile is expected to help improve tolerance and overall treatment outcomes of FLT3-targeted therapy. BMF-500 was designed as a highly potent and selective, covalent, small molecule inhibitor of FLT3, that binds irreversibly to a reactive cysteine in the kinase active site. BMF-500 is a picomolar inhibitor with markedly improved potency over gilteritinib, a reversible inhibitor of FLT3. BMF-500 selectively killed AML cells harboring FLT3 activating mutations, including MV4-11 and MOLM-13, and engineered cells expressing FLT3 internal tandem duplications (FLT3-ITD) and/or FLT3 tyrosine kinase domain (TKD) mutations. In ex vivo cultures, BMF-500 as a single agent induced potent growth inhibition of patient-derived AML cells harboring either FLT3-ITD or FLT3 non-ITD mutations. The potent covalent inhibition of FLT3 by BMF-500 manifested in effective and durable cellular response that was improved over gilteritinib. For example, a 3-hour exposure followed by wash-out of BMF-500 outperformed 4 days of continuous exposure to gilteritinib, at all concentrations tested. In cells harboring FLT3 activating mutations, BMF-500 induced dose-dependent inhibition of FLT3 phosphorylation and downstream signaling, including phospho-STAT5 and phospho-ERK. A 1-hour pulse treatment with BMF-500 was sufficient to achieve deep and durable target inhibition for greater than 24 hours, an effect not observed with gilteritinib under similar conditions. Profiling BMF-500 across a broad panel of kinases and key cell-surface receptors revealed high selectivity for FLT3 mutants and selectivity against cKit. Potent FLT3 inhibition and high selectivity of BMF-500 translated to sustained tumor regression and improved survival in both subcutaneous and disseminated xenograft models of mutant FLT3-driven AML. Orally administered BMF-500 was well tolerated over 4 weeks of dosing. Study results including efficacy and PD response will be presented. Collectively these data demonstrate BMF-500 to be a novel FLT3 inhibitor with best-in-class potential, given its efficacy, durability, and selectivity in comparison to existing FLT3 inhibitors.

Treatment Patterns and Outcomes of Patients with NPM1+ Acute Myeloid Leukemia (AML) Enrolled in the CONNECT® Myeloid Disease Registry

Background Mutations in NPM1 (NPM1+) occur in ∼30% of patients with AML. NPM1+ has been associated with favorable outcomes following intensive chemotherapy (IC) and demonstrated positive prognostic value in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) mutations. Recently, several studies have reported differences in outcomes in patients with NPM1 mutations based on age and treatment. In this analysis we evaluated treatment patterns and outcomes in AML based on NPM1 mutational status and primary induction therapy in patients who were enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011). Methods Patients aged 55 years or older with AML were grouped by known NPM1 status (NPM1+ vs NPM1‒) and induction therapy (non-IC vs IC). Overall survival (OS) and event-free survival (EFS) were evaluated using the Cox model adjusted for age. For the EFS analyses, an event was defined as death, disease progression, or refractory to therapy. Results A total of 483 patients with known NPM1 status were assessed (NPM1+, n = 123; NPM1‒, n = 360). Most patients were male (NPM1+, 61.5%; NPM1‒, 60.7%) and white (NPM1+, 78.0%; NPM1‒, 85.6%; Table). Median age, Eastern Cooperative Oncology Group performance status (ECOG PS), and frailty group were similar in the NPM1+ and NPM1‒ groups. Favorable and adverse European LeukemiaNet (ELN) risk scores at baseline were different in the NPM1+ and NPM1‒ groups. The majority of patients received non-IC regimens as induction therapy (NPM1+, 69.1%; NPM1-, 75.0%). Overall, patients with NPM1+ AML had significantly longer median OS (26 mo) compared with patients with NPM1- AML (15 mo) (HR [95% CI]: NPM1+ vs NPM1- 0.58 [0.44-0.76]; P < 0.01). When analyzed by type of induction regimen (non-IC vs IC), although OS remained numerically longer for patients in the NPM1+ groups, the differences were not statistically significant. Among patients who received non-IC regimens, median OS was 19 and 14 mo in the NPM1+ and NPM1- groups, respectively (Figure). Among patients who received IC regimens, median OS was 24 and 16 mo in the NPM1+ and NPM1- groups, respectively. There was a significant improvement in EFS for patients in the NPM1+ group vs the NPM1- group (20 mo vs 11 mo; hazard ratio [HR] [95% CI], NPM1+ vs NPM1- 0.61 [0.47-0.80]; P < 0.01). This was also true for patients treated with non-IC regimens (15 vs 11 mo in the NPM1+ and NPM1- groups, respectively; HR [95% CI], NPM1+ vs NPM1- 0.67 [0.46-1.00]; P = 0.048). In contrast with the non-IC treated regimens, in patients treated with IC regimens, NPM1+ status was not associated with significantly longer EFS compared to NPM1- status. Patients with known NPM1 status were further evaluated for additional mutations and the following differences were noted: the frequency of mutations in tumor protein p53 (TP53) (1.6% vs 18.6%), RUNX1 (0.8% vs 14.8%), ASXL1 (2.4% vs 17.6%) IDH2 (8.9% vs 15.7%), and NRAS/KRAS (8.9% vs 14.3%) was lower in patients with NPM1+ vs NPM1- AML, respectively, whereas the frequency of mutations in FLT3-ITD (35.8% vs 17.1%), FLT3- tyrosine kinase domain (TKD) (14.6% vs 6.2%), DNMT3A (23.6% vs 15.7%), and IDH1 (14.6% vs 8.6%) was higher in patients with NPM1+ vs NPM1- AML, respectively. Conclusions Overall, patients with NPM1+ AML had longer OS and EFS than patients with NPM1‒ AML. This was observed among patients with NPM1+ AML for both non-IC and IC induction regimens. Patterns of co-mutations differed between the NPM1+ and NPM1- groups and is a likely contributing factor to differences between outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FLT3-ITD Degrades CEBPA Via the Ubiquitin-Proteosome Pathway

FLT3 internal-tandem-duplication (FLT3-ITD) mutations are highly recurrent in acute myeloid leukemias (AML). Although secondary messenger impacts of these receptor tyrosine kinase mutations are well characterized, downstream mechanisms by which myeloid precursors are transformed remain unclear. One clue is that FLT3-ITD and CEBPA bi-allelic mutations are mutually exclusive, suggesting that FLT3-ITD might impact CEBPA, a master transcription factor mandatory for granulo-monocytic lineage-differentiation. In fact, FLT3-signaling has been shown to phosphorylate CEBPA at serine 21 (S21-P), and inhibition of this post-translational modification resumed granulocytic-differentiation of FLT3-ITD AML cells (Radomska et al, J Exp Med 2006). How S21-P post-translational modification of CEBPA blocks granulocyte lineage-differentiation, however, was unknown. To examine FLT3-ITD impacts on CEBPA function in primary AML cells, we examined expression of CEBPA target genes (24 genes identified by CEBPA ChIP-seq, Jakobsen et al, Genome Res 2013): CEBPA target genes positively correlated with CEBPA mRNA expression in normal granulopoiesis and in primary FLT3/CEBPA-wildtype (WT) AML cells (n=112) (Pearson correlation coefficient [ρ] 0.36, p<0.0001), but not in primary FLT3-ITD AML cells (n=35)(ρ =-0.15, p=0.4). Moreover, CEBPA target genes were almost 2-fold less expressed in the FLT3-ITD AML cells (p<0.0009), despite CEBPA mRNA expression comparable to the WT AML cells (RNA-sequencing, TCGA). To investigate the disconnect between CEBPA mRNA expression and CEBPA target gene activation, we analyzed CEBPA protein, and found very low amounts in FLT3-ITD AML cells MOLM13 and MV411 compared to large CEBPA protein amounts detected in WT THP1 AML cells, both by Western blot and immuno-fluorescence, again despite similar CEBPA mRNA expression. This observation complemented published data from others that FLT3-ITD introduction into murine 32D cells suppressed total Cebpa protein (Mizuki et al, Blood 2003; Zheng et al, Blood 2004). The FLT3-inhibitors sunitinib and gilteritinib decreased CEBPA S21-P, increased CEBPA total protein measured by Western blot and immunofluorescence, and induced terminal granulocytic differentiation in the FLT3-ITD MOLM13 and MV4-11 AML cells, but not FLT3-WT THP1 or OCI-AML3 cells. Moreover, the proteosome inhibitors bortezomib or MG132 increased S21-P-, ubiquitinated-, and total-CEBPA protein in the FLT3-ITD but not the FLT3-WT AML cells. We therefore further investigated interactions of endogenous CEBPA with the ubiquitin-proteosome system (UPS): unbiased analyses by immunoprecipitation (IP)-LCMS/MS demonstrated 3-4 fold enrichment of the deubiquitinase USP7 and E3 ubiquitin ligase UHRF1 in the CEBPA interactome of FLT3-ITD versus FLT3-WT AML cells. Reverse analyses of the endogenous USP7 protein interactome by IP-LCMS/MS and IP-Western blot confirmed USP7 interactions with CEBPA and UHRF1 and demonstrated USP7 phosphorylation at serine-18 (S18-P), a candidate for regulation by FLT3. The small molecule P22077 alters and inhibits USP7 catalytic site conformation: P22077 shifted USP7 deubiquitinase interactions from UHRF1 to CEBPA, decreased UHRF1 and increased total CEBPA (Figure). FLT3-ITD AML cells were >2-fold more sensitive to P22077 than FLT3-wt AML cells (IC50 5.8 and 7.1 µM for MV411 and MOLM13 versus 13.65 and 21.7 µM for THP1 and K562 cells) (Figure). Another mystery in FLT3-ITD AMLs is relative chemo-resistance even though p53 is wild-type - USP7 regulates p53, and thus FLT3-ITD regulation of USP7 may contribute to chemo-resistance, a link we are currently consolidating. In sum, FLT3-ITD signals for CEBPA protein degradation via USP7 and UHRF1, to thereby decouple proliferation from onward lineage-maturation in committed granulo-monocytic progenitors. Overall, this pathway explains FLT3-ITD: (i) origin in committed granulo-monocytic progenitors; (ii) link to suppressed CEBPA-target genes; (iii) mutual exclusivity with bi-allelic CEBPA mutations; (iv) differentiation-syndrome upon treatment with FLT3-inhibitors; (v) sensitivity to UPS inhibitors; (vi) mutual exclusivity with TP53-mutations yet chemo-resistance, and can inform understanding of how other receptor tyrosine kinase mutations, e.g., KIT mutations, transform committed lineage-progenitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal