Internal Tandem Duplication Mutations Research Articles

Abstract 1062: A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme

Abstract AML is the most common acute leukemia in adults over 50 years of age. Current therapeutic approaches are focused on high or low dose chemotherapy in the front line setting, with use of molecularly targeted agents in unfit patients or the relapsed/refractory setting. However, routine genomic profiling for common molecular lesions (e.g. FLT3 mutation) incompletely stratifies the target patient population, and more personalized and comprehensive approaches could be of significant clinical benefit. To this end, we have developed an in vitro functional genomics platform for the systematic characterization of gene dependency on drug targets using CRISPR/Cas9 technology. Methods: Retrospective pre-therapy samples were obtained for a cohort of AML patients who subsequently went onto therapy with a combination of the tyrosine kinase inhibitor sorafenib plus chemotherapy (e.g. GCLAM). Unsorted primary tumor cells from these samples were transduced with lentivirus harboring Cas9 enzyme and an sgRNA library. This customized sgRNA library (Fx Heme) was designed to inhibit expression of genes encoding the targets of all FDA-approved oncology drugs, as well as internal positive and negative references. Transduced cells were harvested at multiple timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Dependency on targets of sorafenib was compared to clinical outcome (complete response (CR) without minimal residual disease (MRD), incomplete response, CR with MRD, or refractory) following treatment with sorafenib plus chemotherapy. Results: Among the 22 AML patients analyzed, 14 reached CR and 8 were classified as incomplete responders (refractory or MRD). Gene dependency on sorafenib targets including FLT3, KIT, PDGFR, and all RAF isoforms was frequently observed (12/22 cases). Among cases indicating any sorafenib target gene dependency, 5/12 (42%) demonstrated dependence on 2 or more sorafenib target genes. Overall, Fx Heme achieved 78.6% sensitivity, 87.5% specificity, and 91.7% PPV for prediction of observed clinical outcome. In contrast, stratification based on FLT3 mutation (internal tandem duplication or tyrosine kinase domain mutation) demonstrated 64.3% sensitivity, 50% specificity, 69.2% PPV. In summary, our results establish feasibility of CRISPR-based Fx Heme comprehensive functional genomic profiling for AML precision medicine, and validate this approach in a retrospective study cohort. Functional genomics may contribute to more effective personalization of AML treatment by uncovering drug target dependencies not readily identified by conventional genomic profiling, in an unbiased, comprehensive and patient-specific fashion. Citation Format: Stephane Corbel, Karl Hodel, Kathleen Tran, Maci Meyers, Jing Tong, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath. A comprehensive CRISPR-enabled functional genomics profiling platform in acute myeloid leukemia (AML): Pilot study and validation of Fx Heme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1062.

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.

Sitravatinib as a potent FLT3 inhibitor can overcome gilteritinib resistance in acute myeloid leukemia

BackgroundGilteritinib is the only drug approved as monotherapy for acute myeloid leukemia (AML) patients harboring FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation throughout the world. However, drug resistance inevitably develops in clinical. Sitravatinib is a multi-kinase inhibitor under evaluation in clinical trials of various solid tumors. In this study, we explored the antitumor activity of sitravatinib against FLT3-ITD and clinically-relevant drug resistance in FLT3 mutant AML.MethodsGrowth inhibitory assays were performed in AML cell lines and BaF3 cells expressing various FLT3 mutants to evaluate the antitumor activity of sitravatinib in vitro. Immunoblotting was used to examine the activity of FLT3 and its downstream pathways. Molecular docking was performed to predict the binding sites of FLT3 to sitravatinib. The survival benefit of sitravatinib in vivo was assessed in MOLM13 xenograft mouse models and mouse models of transformed BaF3 cells harboring different FLT3 mutants. Primary patient samples and a patient-derived xenograft (PDX) model were also used to determine the efficacy of sitravatinib.ResultsSitravatinib inhibited cell proliferation, induced cell cycle arrest and apoptosis in FLT3-ITD AML cell lines. In vivo studies showed that sitravatinib exhibited a better therapeutic effect than gilteritinib in MOLM13 xenograft model and BaF3-FLT3-ITD model. Unlike gilteritinib, the predicted binding sites of sitravatinib to FLT3 did not include F691 residue. Sitravatinib displayed a potent inhibitory effect on FLT3-ITD-F691L mutation which conferred resistance to gilteritinib and all other FLT3 inhibitors available, both in vitro and in vivo. Compared with gilteritinib, sitravatinib retained effective activity against FLT3 mutation in the presence of cytokines through the more potent and steady inhibition of p-ERK and p-AKT. Furthermore, patient blasts harboring FLT3-ITD were more sensitive to sitravatinib than to gilteritinib in vitro and in the PDX model.ConclusionsOur study reveals the potential therapeutic role of sitravatinib in FLT3 mutant AML and provides an alternative inhibitor for the treatment of AML patients who are resistant to current FLT3 inhibitors.

MAST CELL NEOPLASIA IN THE GREEN IGUANA, IGUANA IGUANA.

This case series is the first description of mast cell neoplasia in green iguanas, Iguana iguana. All iguanas were adults. Six were females, and four were males. Lesions were associated with the oral cavity in 3 of 10 cases, and on the head in 3 of 10 cases. Three of the cases had masses over the trunk; a single case had a lesion on a limb. Single masses were most common involving the face or oral cavity (6 of 10), whereas four cases with neoplasia distributed along the trunk had multiple masses. One case developed peripheral blood mastocytosis. In most neoplasms, cells contained metachromatic granules in toluidine blue-stained sections. Polymerase chain reaction identified no internal tandem duplication mutation in exon 11 of c-kit, but a mutation in exon 8 in a single case. Compared with other reptiles, green iguanas may be predisposed to development of mast cell neoplasia.

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.

CD44‐targeted chimeric antigen receptor‐T eradicates acute myeloid leukaemic stem cells

CD44‐targeted chimeric antigen receptor‐T eradicates acute myeloid leukaemic stem cells

FLT3 Gene Mutations in Acute Myeloid Leukemia Patients in Northeast Thailand.

BACKGROUND Mutations in the FLT3 gene are associated with acute myeloid leukemia (AML). FLT3 mutations have been identified in approximately 30% of de novo AML patients, particularly those with typical karyotype and inferior prognosis. Therefore, we examined the frequencies of an internal tandem duplication (ITD) and missense mutations of the FLT3 gene and their associated clinical characteristics in patients with AML in northeast Thailand. MATERIAL AND METHODS The leftover bone marrow and/or peripheral blood specimens of 65 newly diagnosed AML patients recruited from Srinagarind Hospital, Khon Kaen University, northeast Thailand, between January 2020 and May 2021 were included in this study. FLT3-ITD and FLT3- tyrosine kinase domain (TKD) were amplified using PCR-related techniques. RESULTS The frequencies of FLT3-ITD and TKD were found to be 16.9% (11/65) and 10.8% (7/65), respectively. One patient had ITD and TKD mutations. The white blood cell count and peripheral blast percentage of FLT3-ITD-positive patients were statistically significantly higher than those of the FLT3-wild type patients, while the peripheral blast percentage of FLT3-TKD-positive patients was significantly lower. No other clinical characteristics among FLT3-positive and FLT3-wild-type patients were observed. DNA sequencing identified 4 FLT3-TKD mutations. The c.2504A>T; Asp835Val and c.2503G>C; Asp835His mutations were predicted as pathogenic mutations while the 2 novel mutations, c.2508C>A; Ile836= and c.2508C>G; Ile836Met were predicted as neutral mutations. CONCLUSIONS This study showed for the first time that FLT3-TKD mutation is common among northeast Thai AML patients. The data should prove useful for selecting efficacious targeted treatment plans for the 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.

Discovery of oxindole-based FLT3 inhibitors as a promising therapeutic lead for acute myeloid leukemia carrying the oncogenic ITD mutation.

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) patients. In the current study, the oxindole chemotype is employed as a structural motif for the design of new FLT3 inhibitors as potential hits for AML irradiation. Cell-based screening was performed with 18 oxindole derivatives and 5a-c inhibited 68%-73% and 83%-91% of internal tandem duplication (ITD)-mutated MV4-11 cell growth for 48- and 72-h treatments while only 0%-2% and 27%-39% in wild-type THP-1 cells. The most potent compound 5a inhibited MV4-11 cells with IC50 of 4.3 µM at 72 h while it was 8.7 µM in THP-1 cells, thus showing two-fold selective inhibition against the oncogenic ITD mutation. The ability of 5a to modulate cell death was examined. High-throughput protein profiling revealed low levels of the growth factors IGFBP-2 and -4 with the blockage of various apoptotic inhibitors such as Survivin. p21 with cellular stress mechanisms was characterized by increased expression of HSP proteins along with TNF-β. Mechanistically, compounds 5a and 5b inhibited FLT3 kinase with IC50 values of 2.49 and 1.45 µM, respectively. Theoretical docking studies supported the compounds' ability to bind to the FLT3 ATP binding site with the formation of highly stable complexes as evidenced by molecular dynamics simulations. The designed compounds also provide suitable drug candidates with no violation of drug likeability rules.

Non-Canonical FLT3 Mutations (FLT3m) in Patients (Pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) - Outcomes and Prognosis after FLT3 Inhibitor (FLT3i) and Venetoclax Based Regimens

Non-Canonical FLT3 Mutations (FLT3m) in Patients (Pts) with Newly Diagnosed Acute Myeloid Leukemia (AML) - Outcomes and Prognosis after FLT3 Inhibitor (FLT3i) and Venetoclax Based Regimens

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

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

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

Gilteritinib Maintenance Therapy Post-Allogenic Stem-Cell Transplantation Improves the Prognosis of Patients with FLT3-Mutated AML

Introduction FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) and -tyrosine kinase domain (TKD) mutation were common mutations in acute myeloid leukemia (AML). FLT3-ITD mutation adversely affected the outcome of hematopoietic stem-cell transplantation (SCT). While sorafenib and midostaurin as maintenance therapy after SCT prolonged relapse-free and overall survival (OS), there are very few reports of post-transplant gilteritinib maintenance therapy outside of clinical trials. Hence, to explore the efficacy of the gilteritinib maintenance therapy post-SCT, we here report the prognostic impact of post-SCT gilteritinib maintenance in a clinical setting. Methods We retrospectively analyzed 25 FLT3-mutated relapsed or refractory AML patients who received allogeneic SCT at our center from January 1, 2011 to April 30, 2022. FLT3 mutation was analyzed by a commonly used polymerase chain reaction (PCR)-based assay. Measurable residual disease (MRD) was assessed by Wilms tumor 1 (WT1) mRNA expression in peripheral blood, and MRD positivity was defined as WT-1 >50 copies/μg in this study. Relapse was defined as an increase of 5% blasts or more. Non-relapse mortality (NRM) was defined as death without relapse. Leukemia-free survival (LFS) was defined as the time from transplantation to leukemia relapse or death from any cause, which comes first. OS was defined as the time from transplantation to death from any cause. Results The median age was 52 years. Twenty-two patients had ITD mutation and 5 had TKD mutation (2 had both ITD and TKD mutation). At the time of the transplant, 20 patients were in remission (6 were MRD-negative and 14 were MRD-positive) and five were in non-remission. All patients achieved hematologic complete remission (CR) after SCT and no patients relapsed within 30 days post-SCT. After hematological engraftment, 14 patients started to receive gilteritinib as maintenance therapy and 11 patients did not. Between the two groups, the patients' characteristics including disease status at transplant, SCT procedures, and donor sources were well balanced except for the administration of FLT3-inhibitor prior to SCT (100% in patients with gilteritinib maintenance and 45.5% in those without gilteritinib maintenance, p = 0.003). Eleven patients (78.6%) in the gilteritinib group and eight (72.7%) patients in the non-gilteritinib group had residual MRD in hematological CR or were non-CR prior to SCT. The median time from SCT to the initiation of gilteritinib was 36 days. The median dose at gilteritinib initiation was 40mg (range 20-120 mg). One patient developed grade 2 skin acute graft-versus-host disease (GVHD) after gilteritinib administration and 4/14 patients (28.6%) developed moderate-severe chronic GVHD (cGVHD, two developed pulmonary cGVHD, one eye and mouse, and one skin) with a median onset of 10.5 months (range 3.1-30.5 months), while only one patient temporary discontinued gilteritinib until the improvement of cGVHD. Late engraftment failure during gilteritinib maintenance was not observed. In terms of survival benefit, patients with gilteritinib maintenance showed significantly longer LFS and OS than those without gilteritinib (1-y LFS; 100% vs. 36.4%, 95% CI; 100-100% vs. 11.2-62.7%, p = 0.0028, Figure A, and 1-y OS; 100% vs. 45.5%, 95% CI; 100-100% vs. 16.7-70.7%, p = 0.0075, Figure B). The cumulative incidence of AML relapse at one-year in patients with gilteritinib maintenance group was significantly lower than those without (0% vs. 51.5%, 95% CI; 0-0% vs. 24.9-86.0%, p = 0.005). Especially, among patients with MRD positive or non-CR prior to SCT (n=19), patients who received gilteritinib maintenance showed a lower cumulative incidence of AML relapse at one-year than those without gilteritinib maintenance (0% vs. 68.8%, 95% CI; 0-0% vs. 35.9-95.2%, p = 0.0028, Figure C). Patients without MRD prior to SCT did not relapse with or without gilteritinib maintenance. These results suggest that gilteritinib maintenance therapy contributes to preventing post-transplant relapse in patients who remain MRD positive at the time of SCT. Conclusion Gilteritinib maintenance therapy post-SCT showed survival benefit in a clinical setting. 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

CD25 Targeting Eliminates Regulatory T Cells and CD25+ Blasts in Acute Myeloid Leukemia

Background Acute Myeloid leukemia (AML) is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype. CD25 Mab (also referred to as RG6292 and RO7296682) is an afucosylated, IL-2 non-blocking human IgG1 antibody, shown to efficiently deplete immunosuppressive regulatory T cells (Tregs) in humans and solid tumour models (Kolben 2021) whilst allowing binding of IL-2 to effector T cells and the induction of anti-tumour adaptive immune responses (Solomon, Amann et al. 2020). CD25 Mab binds to CD25+ target cells and its crystallisable fragment (Fc) to Fc receptors (FcR) expressed on the surface of effector cells, such as FcgRIIIa on Natural Killer (NK) cells, monocytes and macrophages. It mediates killing of target cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). CD25 Mab is currently being investigated in a phase I dose escalation monotherapy study (NCT04158583) or in combination with atezolizumab (NCT04642365). In AML patients, a higher frequency of immunosuppressive bone marrow Tregs was observed as compared to that of healthy donors and their abundance correlated with poorer outcome (Szczepanski, Szajnik et al. 2009, Han, Dong et al. 2018, Dong, Han et al. 2020). In addition, CD25 is also expressed on a subset of AML cells and may be restricted to leukemic stem cells (LSC) or cells with a progenitor phenotype (Angelini, Ottone et al. 2015, Bertolini, Kageyama et al. 2018, Aref, Azmy et al. 2020). Considering the critical role of LSC in the propagation of the disease and the high relapse rate in AML, it would be important to understand if CD25 targeting could deepen responses and help prevent relapse in patients. In this study, we hypothesized that CD25 Mab may have a dual mode of action whereby it depletes suppressive Tregs and has a direct cytotoxic effect on CD25+ AML cells. Methods We conducted an in-depth characterization of bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry (up to 39 cellular markers) and RNA sequencing using computational analysis. Moreover, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab on Tregs and CD25+ AML cells, as compared to isotype control antibody. Results We found that the abundance of Tregs and CD25+ AML cells correlated strongly with that of the blood in 11 patients with time-matched samples, indicating that blood samples could be used for the identification of predictive biomarkers. Moreover, we observed a strong enrichment in the prevalence of CD25 expressing AML cells in patients bearing internal tandem duplication mutations in the FLT3 gene (FLT3-ITD). Furthermore, all four patients treated with a hypomethylating agent in combination with venetoclax displayed detectable levels of CD25+ AML cells. We adopted a patient-centric approach (n=14 patients) to study AML clusters with CD25 expression, due to the high inter-patient heterogeneity. Interestingly, CD25+ clusters most commonly expressed CD34, an LSC marker indicating a preferential expression in immature AML cells. Finally, ex vivo treatment of primary AML patient samples with CD25 Mab led to the specific killing of CD25+ AML cells and Tregs by allogeneic NK cells, as compared to isotype control treatment. Taken together, these results provide a proof of concept of CD25 Mab's dual mode of action in AML patient samples. Conclusions Using high dimensional flow cytometry and computational analysis, we provide a deep characterization of AML patient samples and highlight the heterogeneity in the pattern of expression of common AML targets currently under clinical investigation. We dissected the expression of CD25 on healthy T cells and malignant cells and demonstrated that the blood offers a window into the bone marrow composition and could be used to ascertain Treg and CD25+ AML cells prevalence. Mode of action studies demonstrated that CD25 Mab depletes suppressive Tregs and has a direct cytotoxic effect on the CD25+ AML cells. CD25 targeting represents an attractive target for the treatment of AML, especially in patients where CD25 is expressed on LSC or immature AML cells. Our study warrants further exploration of CD25 Mab as combinatorial treatment with, for instance, FLT3 inhibitors or venetoclax.

Clinical characteristics and prognostic analysis of acute myeloid leukemia patients with PTPN11 mutations

ABSTRACT Objectives Little is known about the clinical impact of germline/somatic mutations of PTPN11 in acute leukemia. The aim of this study was to investigate the clinical characteristics and prognostic impact of PTPN11 mutations in patients with acute myeloid leukemia (AML). Methods: Seventy-four patients with PTPN11 mutation-positive AML treated at our institution were enrolled in this study. The prevalence of PTPN11 mutations was examined using targeted next-generation sequencing technology, and patients with AML and PTPN11 mutations were screened. Clinical characteristics, prognostic impact, and association between PTPN11 mutations and other mutations were analyzed retrospectively. Results: PTPN11 mutations co-occurred more commonly with DNMT3A, NPM1, and FLT3 internal tandem duplication mutations. Compared with PTPN11 wild-type (WT) patients, PTPN11 mutation-positive AML patients presented with higher white blood cell (WBC) and platelet (PLT) counts. In 74 PTPN11 positive AML patients, PTPN11 mutations had an adverse effect on overall survival (OS) (62.5%) and a negative prognostic effect on event-free survival (EFS) (50%). Allo-hematopoietic stem cell transplantation (HSCT) abrogated the negative effect of mutations in PTPN11; the OS and EFS of AML patients with PTPN11 mutations who received transplantation were longer than those of AML patients with PTPN11 mutations who did not undergo allo-HSCT (P = 0.001, EFS; P < 0.001, OS). Discussion: Newly diagnosed PTPN11 mutation-positive AML patients with high WBC and PLT counts or presenting no remission after first induction chemotherapy suffer from high mortality rates. Conclusion: Given the lack of targeted therapies for PTPN11 mutations, timely HSCT is necessary for patients.

AML-326 Novel FLT3-ALK2 Dual Inhibitor TP-0184 Inhibits Leukemia Growth by Targeting Serine Biosynthesis in FLT3-ITD-Positive AML Cells

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) inhibitors have been studied; however, molecular factors contributing to this resistance are unknown. i) To investigate the clinical significance of BMP type 1 receptor ALK2 in AML patients with FLT3-ITD mutations. ii) To elucidate the therapeutic effect of FLT3-ALK2 dual inhibitor TP-0184 on leukemia growth. To determine whether ALK2 is a potential target in FLT3-mutated AML patients, we analyzed OHSU and TCGA datasets. We treated AML cell lines with FLT3-WT or -ITD mutations with ALK2 inhibitors and measured their effect on cell cycle and proliferation. To determine the mechanism of TP-0184, we measured the activation of FLT3 downstream signaling by using western blotting and RNA sequencing. Further, we performed kinase profiling to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, we investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutated PDX and xenograft models. Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (P<0.00001) and predicts poor overall survival (P=0.05). Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50<25 nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100 nM). Interestingly, TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5 and p-ERK, as well as p-PI3K, p-mTOR, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway. Lastly, treatment with TP-0184 significantly prolonged the survival of FLT3-ITD-mutated AML-bearing mice (P<0.0001). Our data indicate that ALK2 is a prognostic marker in FLT3-mutated AML. TP-0184 inhibits cell proliferation by inhibiting FLT3 downstream signaling pathways in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3-ALK2 dual inhibitor. TP-0184 inhibits AML growth in FLT3-mutated PDX and xenograft models.

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.

Antileukemic activity of YPN-005, a CDK7 inhibitor, inducing apoptosis through c-MYC and FLT3 suppression in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive blood cancer with a high rate of relapse associated with adverse survival outcomes, especially in elderly patients. An aberrant expression of cyclin dependent kinase 7 (CDK7) is associated with poor outcomes and CDK7 inhibition has showed antitumor activities in various cancers. We investigated the efficacy of YPN-005, a CDK7 inhibitor in AML cell lines, xenograft mouse model, and primary AML cells. YPN-005 effectively inhibited the proliferation of AML cells by inducing apoptosis and reducing phosphorylation of RNA polymerase II. The c-MYC expression decreased with treatment of YPN-005, and the effect of YPN-005 was negatively correlated with c-MYC expression. YPN-005 also showed antileukemic activities in primary AML cells, especially those harboring FMS-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD) mutation and in in vivo mouse model. Phosphorylated FLT3/Signal transducer and activator of transcription 5 (STAT5) was decreased and FLT3/STAT5 was downregulated with YPN-005 treatment. Our data suggest that YPN-005 has a role in treating AML by suppressing c-MYC and FLT3.