FLT3 Mutations Research Articles

Abstract P09: AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA

Abstract FMS-like tyrosine kinase-3 (FLT3) mutations are often linked with poor prognosis in Acute myeloid leukemia (AML). Despite increasing availability of targeted therapy, such as FLT3 inhibitors (FLT3i), remission is often short-term and followed by relapse and drug resistance. With varying response rates to standard treatments, the need to understand the molecular mechanisms driving drug resistance and accurately predict responses to treatment is crucial. Here, we present the ability of Quadratic phenotypic optimization platform (QPOP), in accurately identifying potential responders to possible combination therapies in AML. AML cell lines and AML patient samples were treated with a set panel of drugs including FLT3i. QPOP drug combination scores and ranking were derived from cell viability response following treatment. 40 patients were recruited for the study, of which 20 patients were evaluated for their clinical concordance. RNA sequencing and molecular analysis was further conducted to determine the accuracy of QPOP predicted response and to determine mechanism of resistance to FLT3i. QPOP predicted response was concordant to patient treatment outcome with 80% sensitivity and specificity. QPOP analysis on AML cell lines accurately identified responders to FLT3i including the differential response between a FLT3- ITD isogenic parental and FLT3i resistant cell line. Serial QPOP analysis in a FLT3 mutant patient sample indicated a decrease in FLT3i sensitivity concordant with an increasing FLT3 allelic burden and the development of resistance. The development of resistance to FLT3i based combinations was correlated to an increase in phosphorylated AKT expression. Activation of AKT is associated with the upregulation of Forkhead box M1 (FoxM1) in the resistant cell line. Co-treatment with an AKT inhibitor restored sensitivity to FLT3i treatment in the resistant cells. We demonstrate that QPOP analysis accurately predicts sensitivity to drug-combinations and can be used as a tool to identify sensitivity to FLT3i-based combination therapy in AML. Citation Format: Noor Rashidha Bte Meera Sahib, Jen Wei Ying, Melissa Ooi, Yen Lin Chee, Jayalakshmi, Jameelah Bte Sheik Mohamed, Masturah Rashid, Edward KH Chow. AN EX-VIVO COMBINATION DRUG SENSITIVITY PLATFORM FOR PREDICTING SENSITIVITY TO FLT3 INHIBITOR-BASED COMBINATION IN ACUTE MYELOID LEUKEMIA [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P09.

Structure-Based Optimization of Pyrazinamide-Containing Macrocyclic Derivatives as Fms-like Tyrosine Kinase 3 (FLT3) Inhibitors to Overcome Clinical Mutations.

Secondary mutations in Fms-like tyrosine kinase 3-tyrosine kinase domain (FLT3-TKD) (e.g., D835Y and F691L) have become a major on-target resistance mechanism of FLT3 inhibitors, which present a significant clinical challenge. To date, no effective drugs have been approved to simultaneously overcome clinical resistance caused by these two mutants. Thus, a series of pyrazinamide macrocyclic compounds were first designed and evaluated to overcome the secondary mutations of FLT3. The representative 8v exhibited potent inhibitory activities against FLT3D835Y and FLT3D835Y/F691L with IC50 values of 1.5 and 9.7 nM, respectively. 8v also strongly suppressed the proliferation against Ba/F3 cells transfected with FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD-F691L, FLT3-ITD-D835Y-F691L, and MV4-11 acute myeloid leukemia (AML) cell lines with IC50 values of 12.2, 10.5, 24.6, 16.9, and 6.8 nM, respectively. Furthermore, 8v demonstrated ideal anticancer efficacy in a Ba/F3-FLT3-ITD-D835Y xenograft model. The results suggested that 8v can serve as a promising macrocycle-based FLT3 inhibitor for the treatment of AML.

NGS of brush cytology samples improves the detection of high-grade dysplasia and cholangiocarcinoma in patients with primary sclerosing cholangitis: A retrospective and prospective study.

Biliary dysplasia, a precursor of cholangiocarcinoma (CCA), is a common complication of primary sclerosing cholangitis. Patients with high-grade dysplasia (HGD) or early CCA who have received oncological treatment are candidates for liver transplantation. The preoperative diagnosis of CCA or HGD is challenging, and the sensitivity of biliary brush cytology (BC) is limited. By using next-generation sequencing (NGS), we retrospectively analyzed archived tissue samples (n=62) obtained from explanted liver tissue and CCA samples to identify oncogenic mutations that occur during primary sclerosing cholangitis carcinogenesis. BC samples were prospectively collected from patients with primary sclerosing cholangitis (n=97) referred for endoscopic retrograde cholangiography to measure the diagnostic utility of NGS combined with BC compared with traditional cytology alone. Mutations in KRAS, GNAS, FLT3, RNF43, TP53, ATRX, and SMAD4 were detected in archived CCA or HGD samples. KRAS, GNAS, TP53, CDKN2A, FBXW7, BRAF, and ATM mutations were detected in prospectively collected brush samples from patients with histologically verified CCA or HGD. One patient with low-grade dysplasia in the explanted liver had KRAS and GNAS mutations in brush sample. No mutations were observed in brush samples or archived tissues in liver transplantation cases without biliary neoplasia. While KRAS mutations are common in biliary neoplasms, they were also observed in patients without biliary neoplasia during surveillance. In summary, NGS of BC samples increased the sensitivity of detecting biliary neoplasia compared with traditional cytology. Performing NGS on BC samples may help diagnose HGD or early CCA, benefiting the timing of liver transplantation.

Abstract 5956: FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients

Abstract Backgroud: The prognosis of B cell ALL patients (pts) relapsing after Hematopoietic Transplant is poor and new drugs are needed. In acute myeloid leukemia, FLT3-inhibitors (FLT3i) are showing promising results in FLT3-mutated (mut) cases. Their potential use in FLT3-mut B-ALL has been poorly investigated. Aim: To assess if FLT3 could be a valid targetable marker in B-ALL. Methods: We sequenced with a capture based large RNA-seq panel (1385genes) 183 adult B-ALL [n=52 Pos for Ph or t(4;11) or t(1;19) and n=131 Triple-Negative (TN) pts that are negative for Ph (n=43), t(4;11) and t(1;19) translocations] and 15 donors. To confirm FLT3 muts, 13 available samples were further sequenced with Extended Myeloid Solution (98 genes; SOPHiA) where FLT3 gene is fully covered. In vitro studies using 5 FLT3i [Gilteritinib (Gil), Midostaurin, Crenolanib, Sorafenib and Quizartibin] and Venetoclax (Ven) were conducted on pt primary cells and on TN and other B-ALL cell lines (MUTZ5, MHH-CALL4 Ph-like; NALM6;RS4;11, 697, REH) and 2 AML cell lines (OCI-AML3 FLT3;MV-4-11 FLT3-ITD). Results: We found 15 FLT3 mut in 14/131 TN B-ALL and in 1/43 Ph+ cases: 43.8% were TKD mut, 3 ITD mut, 3 splicing site mut,1 N-terminal, 1 juxta-membrane domain and 1 Ig-like site (Fig. 1A). After pathogenicity interpretation we excluded 3 variants and one mutation was not confirmed at DNA level. Overall 7.6% of TN were mut (Ph+ 2.3%). 81.8% of FLT3mut pts potentially druggable with FLT3i, mainly TKD (63.3%). Targeted NGS revealed the co-occurrence with other mutations (range 2-15), and in particular with CSMD1 (n=4), KDM6A, KMT2D and CREBBP (n=3) alterations. Moreover, 11/13 samples carried copy number alteration (mean 12), mostly amplifications. CDKN2A & IKZF1 were the most frequently altered genes (n=5;4 CN-loss). FLT3 expression was increased in 11/15 cases compared to donors and wt pts. As reported, the expression was higher in ZNF384 or MLLr B-ALL subtypes.To evaluate the effect of FLT3is in ALL, we treated ex vivo primary leukemic cells of 6 adult ALL patients (FLT3-mut n=4; FLT3-wt n=2) with increasing concentrations of 5 FLT3i for 24, 48 and 72h. We observed a trend towards greater response of FLT3-mut ALL cells compared to wt ones. The average IC50 values in response to the five FLT3i was 1.6 uM and 24.5 uM after 72h of treatment in FLT3-mut and FLT3-wt B-ALL, respectively (Fig. 1B). We than evaluated the sensitivity to the 5 Inhs in all cell lines. Interestingly MHH-CALL4 wt were more sensitive to Gil compared to OCI AML3 wt, and showed the same effect compare to MV-4-11 AML mut (F.1C). Given the effect of Gil in single agent, we combined it with Ven in wt B-ALL cells at increasing concentrations. Interestingly We noticed a strong additive effect with the higher Gil concentration and all Ven doses(F.1D). Conclusions: FLT3 alterations identify a novel subgroup of TN B-ALL with therapeutic potential also in combination regimens. Ricerca Corrente Italian MoH L3P1946. Citation Format: Anna Ferrari, Andrea Ghelli Luserna di Rorà, Federico Lo Schiavo, Eugenio Fonzi, Giorgia Capirossi, Chiara Salvesi, Maria Teresa Bochicchio, Lorenzo Ledda, Chiara Servilli, Matteo Paganelli, Giovanni Marconi, Cristina Papayannidis, Chiara Sartor, Michela Rondoni, Anna Maria Mianulli, Barbara Giannini, Crescenza Pasciolla, Fabio Giglio, Sara Galimberti, Monica Fumagalli, Prassede Salutari, Valentina Gaidano, Giovanni Martinelli, Giorgia Simonetti. FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5956.

Abstract 3115: Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform

Abstract Acute myeloid leukemia (AML) is a common hematological malignancy found in adult patients, and FMS-like tyrosine kinase 3 gene (FLT3) mutation occurs in about 30% of patients. In 2019, a phase 3 clinical trial suggested that gilteritinib treatment resulted in significantly longer survival than salvage chemotherapy in relapsed or refractory FLT3-mutated AML patients. Targeting FLT3-mutants in AML has become one of the important therapeutic strategies in this decade. The ex vivo hematological VitroScreen platform provides a powerful tool to evaluate the performance of FLT3 inhibitors and other test articles. With Champions Oncology’s primary hematological repository (https://www.championsoncology.com/hematological-vitroscreen) and proprietary LUMIN Bioinformatics platform, we classified models based on patient responses, clinical annotation, NGS data, and cell surface markers. In this Ex vivo study, we tested gilteritinib in 30 AML patients, and 8 of models were identified as FLT-3 mutants’ carrier. Fold changes of cell viability after gilteritinib treatment were assessed based on quantitation of the ATP present. FLT3-mutants models showed significantly higher sensitivity to gilteritinib than FLT3-WT models (IC50, 132.56 nM vs. 234.16nM) in a 6-day assay. Only 25% of models in the FLT3-mutants group had higher IC50 than the average of all AML models (n=28, IC50 = 204.05 nM), compared to 53% in the FLT3-WT group. Our preclinical data highly match the observations in previous clinical trials, which suggest that VitroScreen is a reliable high throughput tool for drug development of drug resistant cancers. Citation Format: Fu-Ju Chou, Stefano Cairo, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Systematic analysis of gene mutations and therapeutic efficacy via ex vivo hematological vitroscreen platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3115.

Abstract 7153: PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of acute myeloid leukemia

Abstract FLT3 mutations are one of the most frequent genetic alterations in AML, resulting in poor prognosis. Although the development of FLT3 tyrosine kinase inhibitors (TKIs) provided new treatment options, patients with FLT3-mutant AML still suffer from high relapse rates when treated with FLT3 TKI alone. Accumulating evidence supports a combination treatment strategy to lower the recurrence rate. We have previously shown that PHI-101 demonstrated potent activity in various FLT3 mutant AML cell lines when used alone. Here we explore the use of PHI-101 in combination with other therapeutics in preclinical models of AML. We investigated the preclinical efficacy of the combination of PHI-101 with FDA-approved AML therapeutic agents in FLT3 mutant (MV4-11, Molm13, Molm14) and wild-type (THP-1) AML cell lines. Cell growth inhibition was assessed by CCK-8 and/or CellTiter-Glo assays. The Chou-Talalay method was used to calculate the combination index (CI) to test for synergy. Western blotting analysis was performed to explore the potential mechanisms underlying the combination effects. AML xenograft mouse models were used to assess the in vivo efficacy of PHI-101 combination therapy. Statistical comparisons included unpaired t-tests or one-way ANOVA. Combination treatment of PHI-101 with venetoclax or azacytidine showed strong synergistic effects on growth inhibition against both FLT3-ITD mutant (CI<0.3) and wild-type AML cells (CI <0.6). It is noteworthy that the FLT3-ITD homozygous cell line (MV4-11) showed especially strong synergism (CI <0.1). In the xenograft mouse model experiments, 14-day treatment with either PHI-101 alone or in combination with venetoclax or azacytidine showed significant suppression of tumor growth compared to venetoclax or azacytidine alone (in average tumor growth inhibition (TGI) 88.3% vs 8.6%)(p<0.05). In particular, PHI-101 3 mg/kg/day (mpk) with venetoclax 80mpk resulted in a stronger anti-tumor effect (TGI 94.2%) compared to PHI-101 (TGI 87.4%) or venetoclax alone (-9.6% of TGI) (p<0.05) even 21 days after stopping the treatment. In addition, PHI-101 treatment showed a survival advantage in AML animal models, and the combination with venetoclax led to significantly longer survival compared to the control groups. Our preclinical data showed that the combination of PHI-101 with venetoclax or azacytidine resulted in a highly effective anti-leukemic response against AML cells by inducing DNA damage, BCL-2 inhibition, and MCL-1 degradation. Additionally, treatment of mice with low-dose PHI-101 inhibited tumor regrowth and further potentiated venetoclax or azacytidine response in vivo. Our results suggest that the combination of PHI-101 and venetoclax or azacitidine may have the advantage of improving clinical responses in FLT3-ITD AML patients and offers a potential therapeutic option to treat patients with newly diagnosed or relapsed/refractory FLT3-mutant AML. Citation Format: Hee-sun Hwang, Gi-Jun Sung, Kyung-Ah Kim, Ky-Youb Nam, Kyu-Tae Kim, June H. Han, Jeong-Hyeok Yoon, Bao Nguyen, Li Li, Tessa Seale, Donald Small. PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7153.

Abstract 3626: Day 21 early bone marrow response does not offer high concordance with response bone marrow in the induction chemotherapy setting of FLT3 mutant acute myeloid leukemia

Abstract Purpose: Day 14 early bone marrow biopsies are routinely initiated to determine need for reinduction chemotherapy, but poorly predict true response and risk overtreatment. Day 21 (D21) bone marrow biopsies have been performed in the context of trials utilizing FLT3-targeted therapies in combination with induction chemotherapy (Stone et al. NEJM 2017). However, the utility of D21 bone marrow responses is not well defined. Methods: We retrospectively identified 26 newly diagnosed FLT3 mutant AML patients who underwent induction chemotherapy with midostaurin and Next Generation Sequencing (NGS) of bone marrow aspirate within a week of diagnosis. Results: 19/26 (73%) of patients had both D21 and response bone marrow biopsy. Of the remaining 7 patients, 4 patients did not have D21 bone marrow, 2 died of unrelated complications prior to response bone marrow, and 1 did not have recovery marrow available for assessment. Our cohort included 13 (50%) men with a median age of 57 years (range, 21 - 73 years). All patients had ECOG of 0 - 1. 96% of cases were de novo AML and 81% had normal cytogenetics. FLT3 ITD mutations were detected in 17 (65%) patients with a median FLT3 ITD ratio of 0.305 (range, 0.03 - 1) and median FLT3 VAF of 23.8% (range, 3.2 - 80.6%). 7 patients (26.9%) underwent additional allogeneic stem cell transplant. In the 19 patients evaluable for analysis, 13/13 (100%) patients with blast % ≤ 5 at D21 achieved a complete response (CR) based on recovery marrow. 4/6 (67%) patients with blast % > 5 at D21 which were considered to have residual disease based on pathology review (median blast % 11 (range, 6 - 17%) ultimately achieved a CR. The 2 patients that did not achieve a CR has a blast % of 29% and 9% on D21 marrow. Thus, the positive predictive value of D21 early bone marrow response corresponding with the response bone marrow was only 33% in our patient cohort. Of the 4 patients that did not have D21 marrow but had recovery marrows available for review, the CR rate was 100%. No other significant differences in initial disease characteristics were seen. There were no differences proportion of co-mutations in NPM1, DNMT3A, TP53, IDH2, and IDH1, and higher FLT3 ITD ratio nor FLT3 VAF corresponded with ability to clear D21 bone marrow. Additionally, there were no differences in minimal residual disease (MRD) and overall survival (OS) between the groups. Conclusions: Our study suggests early bone marrow response in the context of FLT3-mutant disease is not dependent on inherent disease characteristics and does not correspond to overall response to targeted induction chemotherapy. The utility of these marrows in is this population needs to be studied further in a larger cohort to better understand the clinical necessity to avoid unnecessary cost/procedures to patients and even more importantly unnecessary reinductions. Citation Format: Vincent Lok, Su Bin Hahn, Nikesh N. Shah, Daniel A. Kerr, Ivan M. Borrello, Eduardo M. Sotomayor, David M. Swoboda. Day 21 early bone marrow response does not offer high concordance with response bone marrow in the induction chemotherapy setting of FLT3 mutant acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3626.

Abstract 1966: The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia

Abstract In approximately 23% of AML patients, Fms-like tyrosine kinase 3 (FLT3) contains a gain-of-function, internal tandem duplication (ITD) mutation that is associated with an unfavourable prognosis. FLT3 is therefore seen as a promising therapeutic avenue for AML and as a result, a more complete understanding of its function and signalling may lead to additional targets and resistance mechanisms being identified. To date, several FLT3 inhibitors including Quizartinib (AC220), a second-generation ITD-selective tyrosine kinase inhibitor, have been developed, but single-agent clinical trials have not been overwhelmingly successful. In most preclinical studies, the inhibitory effects of FLT3 inhibitors are mainly evaluated using mutant-expressing models, however, most AML patients harbour both a wild-type and mutant FLT3 allele as well as presenting with high plasma levels of FLT3 ligand (FLT3L). We hypothesized that FLT3L could act through WT-FLT3 to influence the efficacy of FLT3 inhibitors in cells with heterozygous mutations. In this study, we have examined the role of FLT3 inhibition and FLT3L activation on the cellular localisation and downstream signalling in several AML cell lines, some of which express ITD mutations. We also looked at mechanisms by which FLT3L could impair the efficacy of FLT3 inhibitors. Our results revealed that the majority of FLT3 in the MV4-11 and MOLM-13 cells was intracellular. Inhibition of ITD-FLT3 with quizartinib led to a dramatic relocalisation of FLT3 to the cell surface. This effect was more pronounced in MV4-11 (FLT3ITD/ITD) than the heterozygous MOLM-13 (FLT3ITD/WT) cells that also express WT. This was accompanied by inhibition of ERK, AKT and STAT5 signalling pathways and resulted in cell death. Quizartinib induced cell death only in AML lines expressing FLT3-ITD mutations (MOLM-13 and MV4-11), and this could be antagonised by FLT3 ligand (FLT3L). The largest inhibitory effects were seen in heterozygous cells expressing a mutated and wild-type allele. FLT3 inhibition was associated with downregulation of the anti-apoptotic protein Mcl1 and upregulation of the pro-apoptotic BH3-only protein, Bim. Our experiments indicated that both of these proteins were regulated by both WT and ITD-FLT3 through the MAPK pathway. These results suggest that activation of FLT3 signalling by FLT3L confers resistance to quizartinib through upregulation of Mcl-1 and suppression of Bim expression. Taken together, our data suggest that FLT3 cell surface localisation and expression are controlled by ITD mutations and are key components of drug resistance. The data also suggests a novel therapeutic approach in patients with high plasma FLT3L levels, especially when using type II inhibitors such as quizartinib, is to co-target the MAPK/ERK pathway to abrogate the FLT3Lmediated resistance in FLT3-ITD AML. Citation Format: Taha Alqahtani, David MacEwan. The FLT3 tyrosine kinase receptor ITD mutation controls its expression and drug resistance in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1966.

FLAG/FLAG-Ida Regimen in Secondary and Relapsed/Refractory Acute Myeloid Leukemia-Even in the Era of New Treatment Modalities Still a Significant Player.

(1) Background: Relapsed/refractory (r/r) and secondary acute myeloid leukemia are highlighted by chemoresistance and poor outcomes. The aim of the study was to assess the efficacy and toxicity of fludarabine, cytarabine, and granulocyte-colony stimulation factor (FLAG) with or without idarubicin (-Ida) and to discuss novel therapies in this setting. (2) Methods: Clinical and cytogenetic data on 130 consecutive patients with r/r and secondary AML treated at our center were retrospectively analyzed. (3) Results: There were 48, 56, and 26 patients with relapsed, refractory, and secondary AML, respectively. The median age was 60 years. The overall response was achieved in 70% of patients. The median overall survival (OS) time for the whole group was 9.4 months. In total, 47% of patients proceeded to allogeneic hematopoietic stem cell transplantation (aHSCT) and these patients had significantly prolonged OS compared to the others (63 months vs. 4.2 months; p < 0.001). Among the variables, including age, FLT3 mutation status, European LeukemiaNet (ELN) 2022 classification risk, FLAG vs. FLAG-Ida, and aHSCT, a multivariate analysis revealed that only aHSCT significantly influenced overall survival. (4) Conclusions: FLAG(-Ida) chemotherapy remains an effective salvage chemotherapy for patients with r/r and secondary AML with a plan of proceeding to aHSCT.

Abstract 2827: Characterization of acute myeloid leukemia PDX models with hotspot gene mutations for therapeutic evaluation

Abstract Introduction: Acute myeloid leukemia (AML) is the most common acute type of leukemia in adults characterized by chromosomal abnormalities and gene mutations. While inhibitors have shown promise targeting FLT3 mutations, resistance to these inhibitors can emerge. Similarly, inhibitors targeting IDH1 and IDH2 mutations showed promise, yet the emergence of drug resistance poses a significant challenge. In this study, a panel of AML PDX models with multiple gene alterations was established to support the development of new therapies. Methods: The systemic AML PDX models were established using patients’ peripheral blood (PB) or bone morrow (BM) injected intravenously and expanded in vivo by using splenic tumor cells. One AML PDX model derived from patient skin was established subcutaneously. Expression of human CD45+ in PB cells for systemic models or tumor volume for the subcutaneous model was used to monitor the tumor burden. Leukemic loads (hCD45+ cells) were measured in PB, spleen (SP), and BM. Tumors were categorized following tumor immunophenotyping, histology, and RNA sequencing/Whole Exome Sequencing. For in vivo efficacy studies, animals were grouped 3-5 weeks after systemic tumor cells inoculation and treated with AC220 (1, 2 or 10 mg/kg, p.o., QD), Cytarabine (Ara-c, 2mg/kg, i.p., QD), 5-azacytidine (2mg/kg, i.p., QD), Sorafenib (10mg/kg, i.p., QD) and Gilteritinib (10 or 30mg/kg, p.o., QD). Results: Nine systemic and one subcutaneous AML PDX models carrying hotspot mutations (e.i. FLT3-ITD or TKD, IDH1-R132H and IDH2-R140Q) were established, showing comparable features to the clinic. Systemic AM8231 PDX model harboring FLT3-ITD mutation treated with AC220, a type II FLT3i, showed significant tumor burden reduction in PB at different time points and in SP and BM at termination, as well as a survival increase. Similarly in systemic model AM7577 with FLT3-ITD mutation treated with AC220, significant tumor burden reduction in PB was observed. Whereas using systemic model AM9626 (model with Sorafenib pretreatment history) with coexisting-FLT3-ITD/D835H TKD mutation, no significant efficacy was observed with AC220i, suggesting a FLT3-TKD mediated resistance mechanism. To overcome the resistance, Gilteritinib, a type I FLT3i, was tested in AM9626 and showed significant reduction of hCD45+ cells in PB compared to the control group, even at a lowest dose. As model AM9626 PDX also carries IDH1 mutation, the level of serum 2-HG correlated with the tumor burden. The functional effect of IDH1 inhibitor on the tumor inhibition and 2-HG level is under investigation. Conclusion: The results presented in this study showed the establishment of a panel of AML PDX models, including different subtypes and genomic features reflecting the clinic. Those models will support the preclinical evaluation of new treatments or combination modalities as well as a better understanding of drug resistance mechanisms. Citation Format: Qingzhi Liu, Jinxi Wang, Jinping Liu, Likun Zhang, Ludovic Bourre, Jingjing Wang. Characterization of acute myeloid leukemia PDX models with hotspot gene mutations for therapeutic evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2827.

Abstract 611: Discovery of LNX231, a multi-kinase inhibitor mainly target FLT3 with robust in vivo efficacy in FLT3 mutant AML models

Abstract LNX231 is a multi-kinase inhibitor mainly target FLT3, which could both directly kill cancer cells and promote tumor immunity. LNX231 potently inhibits FLT3 activity in biochemical assay, with a 50% inhibitory concentration (IC50) of 3.5 nM, significantly inhibits FLT3-D835Y and FLT3 ITD, the values of IC50 are 20 nM and 4.4 nM. LNX231 inhibits the proliferation of FLT3 mutant AML cell lines via RAS-MARK and JAK-STAT pathway. LNX231 significantly decreases the phosphorylation of STAT5 and ERK in MV-4-11 cells at the high concentration, but has no effect on the phosphorylation of FLT3 and AKT. LNX231 significantly decreases the phosphorylation of FLT3, STAT5 and ERK in MOLM-13 cells at the high concentration, but has no effect on the phosphorylation of AKT. In FLT3 mutated AML models, LNX231 induced sustained tumor shrinkage as a single agent and was well tolerated. At the same dose, LNX231 was more potent than the FDA-approved drug Gilteritinib. In the MC38 tumor-bearing model, the efficacy of LNX231 was better than Sunitinib and Lenvatinib. LNX231 has good oral bioavailability in different species, such as mouse, rat, dog and monkey. DMPK data predicted oral QD dosing in human. It also showed excellent ADMET profile as a pre-clinical candidate. Safety tolerability studies were conducted in mice and dogs. LNX231 has low cardiotoxicity (IC50 of hERG is 25.84 µM) and 5-6 times safety window. In conclusion, we discovered LNX231 as a multi-kinase inhibitor for FLT3 mutated AML. Citation Format: Lizhi Zhao, Guizhou Hao, Rong Deng, Naihua Zhang, Jingchun Yao, Anguo Zhu, Guangyan Li, Enli Wang, Zhenyu Li, Zhong Liu, Guimin Zhang. Discovery of LNX231, a multi-kinase inhibitor mainly target FLT3 with robust in vivo efficacy in FLT3 mutant AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 611.

Loss of Blood Group Antigens in Haematolymphoid Malignancy: A Case Series from a Cancer Institute from North East India

Introduction: Altered expression of blood group antigens has been reported to be associated with both solid as well as haematological malignancies. This usually results from genetic mutation leading to incomplete as well as abnormal synthesis of antigens. Transitional loss of RBC antigens especially from ABO system is most commonly found in haematological malignancies causing blood group discrepancies and followed by reversal to its historical blood group during remission. Loss of A, B antigens has also been associated to play a role in lung, bladder and colon cancers. However not much data is available regarding haematological malignancies.Methods and Methodology: Retrospective review of blood group discrepancy (Forward group) in patients admitted for haematolymphoid malignancy at our institute was done during a period of 3.5 years (13/3/2020 till 12/9/2023). Blood grouping done by Column agglutination test were repeated by conventional tube method. Further confirmation of the group was done by saliva test and adsorption elution test as indicated. The detail history of the patient was collected from the hospital records and analysed. Results: A total of 7 patients presented with either loss or decrease in antigen expression. Most common diagnosis was Acute Myeloid Leukaemia (AML) with FLT3 mutation and most common antigen affected was A. Conclusion: Haematolymphoid malignancies has been associated with loss of blood group antigens causing group discrepancy. Blood transfusion is an integral part of the supportive care in these patients. Hence, proper work up of blood group discrepancy cases should be done and recorded so as to prevent delay in blood transfusion.

Unpredicted transformation of acute myeloid leukemia with translocation (16;16) (p13; q22): a case report and review of the literature

IntroductionThe transformation of acute myeloid leukemia with translocation (16;16) (p13; q22) from AML M2 to acute monocytic leukemia (AML M5) during therapy is a rare clinical occurrence, and this is the first time it has been reported.Clinical complainA 19-year-old male patient was admitted for severe fatigue with anemic manifestation and weight loss, for more than 1 month, with exacerbation of the condition in the last 2 days.DiagnosisA primary diagnosis was made for AML M2 with t (16;16) (p13; q22) established on bone marrow (BM) morphology. A consequential detection of FLT-3 ITD mutation was done. At day 28 follow-up after induction and maintenance therapy, the diagnosis of AML M2 was maintained with a high bone marrow (BM) blast count, prompting the initiation of a more aggressive treatment protocol. After 1 month of implementing the recent protocol, the patient remains morphologically resistant with a notable transformation of bone marrow infiltration by an abnormal monocytic population (monoblasts and promonocytes). The final diagnosis of transforming FLT3-mutated AML with t (16;16) (p13; q22) was established.InterventionAfter the initial diagnosis of AML M2 with t (16;16) (p13; q22), the patient received the 3 + 7 induction protocol. The 2nd induction protocol initiated after the second evaluation and morphological resistance was the FLAG Adrian protocol. The 3rd protocol after transformation to AML M5 was 1 cycle of the MEC protocol. Anti-FLT3 treatment was considered.OutcomesThe patient was maintained on the 3rd protocol of chemotherapy. Unfortunately, he was admitted to the ICU unit complaining of neutropenic fever and severe sepsis where he died before final re-evaluation and the anti-FLT3 treatment initiation.ConclusionAML with t (16;16) (p13; q22) characterized by favorable outcome. However, identifying additional chromosome abnormality or genetic aberration, especially FLT3 gene mutation, is recognized as an important factor influencing final disease outcome. Therefore, early detection of FLT3 mutations will allow comprehensive disease course prediction and targeted therapy that might achieve longer and more durable remissions.

Abstract B102: Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence

Abstract Circulating tumor DNA (ctDNA) is the cancer-derived fraction of extracellular DNA fragments present in the bloodstream as cell-free DNA (cfDNA). Consequently, ctDNA harbors the distinct genomic information specific to the cancer from which it derives. Therefore, ctDNA from liquid biopsies provides a non-invasive, universally available alternative to traditional tumor biopsies. ctDNA amount may provide insight into tumor burden, as its concentration is typically highest during diagnosis and relapse. The non-invasive nature of this method facilitates frequent monitoring, enabling longitudinal tracking of the disease's trajectory and response to treatments. By focusing on the key genomic changes, either short mutations or copy number aberrations (CNA), liquid biopsy allows the investigation of cancer progression and evolution over time, providing invaluable insight into the behavior and adaptability of the cancer. We collected 152 longitudinal plasma samples and 92 fresh tissue samples from 29 patients enrolled in the DECIDER cohort (NCT04846933), after a diagnosis of high-grade serous carcinoma (HGSC), the most common and deadliest histological subtype of ovarian cancer. With ultra-deep targeted sequencing of a gene panel of more than 700 cancer-related genes we were able to comprehensively evaluate the cancer-related genomic changes. The concordance rate between mutations in plasma compared to tumor tissue was 83 % at diagnosis and 90 % at relapse, confirming the reliability of exploiting ctDNA in monitoring genomic alterations. Furthermore, by comparing plasma- and tissue-derived mutations, we demonstrated that comparable levels of ctDNA are released from adnexa, intra-abdominal lesions, and ascites, proposing that a liquid biopsy represents mutations from various locations. Analysis of CNA showed that our approach successfully detected concordant profiles in most plasma samples, even with tumor content as low as 3%. Additionally, highly amplified regions were identified in samples with approximately 1% tumor content. Longitudinal analysis of both mutations and CNA revealed genomic changes in plasma samples at relapse. Twenty-four new exonic mutations were identified in eight patients at relapse; two of these alterations were related to HRD status, supporting PARP-inhibitors as a valid treatment option. Furthermore, mutations in FGFR3, JAK2, FLT3 and CDKN2A could be investigated as target indications for extended use of currently available therapies. Eleven patients had high tumor content in their plasma samples at the time of relapse. We detected altered CNA profiles in seven of these eleven patients, so that five patients harbored only a few novel focal amplifications or losses, while two patients showed significant changes in their copy-number profile during the therapy. In conclusion, our study identified recurrence-specific genomic aberrations, proposing that ctDNA more accurately represents the metastatic tumor and provides possibilities for personalized therapy options. Citation Format: Giovanni Marchi, Mai T.N. Nguyen, Anna Rajavuori, Taru Muranen, Kaisa Huhtinen, Sinikka Oksa, Sakari Hietanen, Johanna Hynninen, Jaana Oikkonen, Sampsa Hautaniemi. Mutational and copy number-based ctDNA profiles mirror high-grade serous cancer tumors and enable detection of genetic changes appearing at recurrence [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B102.

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P &amp;lt; 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Causes and risk factors for early death in adult patients with acute promyelocytic leukemia: a real-life experience

IntroductionEarly Death (ED) remains challenging in newly diagnosed acute promyelocytic leukemia (APL), especially in developing countries. The clinical and laboratory profile at diagnosis were evaluated and causes and risk factors were investigated in adult APL patients. MethodA retrospective real-life analysis of 141 medical records was performed of patients diagnosed with APL between 2007 and 2018, whether they were treated with the IC-APL 2006 protocol or not. Risk factors were assessed by univariate and multivariate analysis. Main resultsOverall, 112 patients were included in the study. ED occurred in 22.3% of cases, surpassing clinical trial reports, with non-protocol-eligible patients presenting notably higher rates (60%), potentially due to their clinical status. Hemorrhage (60%) and infection (33.3%) were the leading causes of ED. Univariate analysis associated ED to the ECOG score; white blood cell (WBC) count; body mass index; levels of hemoglobin, albumin, uric acid, and creatinine, aPTT and INR and FLT3 mutations. Multivariate analysis identified ECOG score ≥2 and elevated WBC count as independent risk factors. ConclusionED remains a substantial challenge in APL, especially in real-world settings with hemorrhage and infection being the leading causes. ECOG status and WBC count emerged as independent risk factors, while age and platelet count lacked a 30-day prognostic correlation. Evaluating prognostic enhancement tools in controlled trials and real-life settings is pivotal to improving APL outcomes.

Leukemic mutation FLT3-ITD is retained in dendritic cells and disrupts their homeostasis leading to expanded Th17 frequency.

Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish bone fide DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet- phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4+ T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4+ T cells in the bone marrow and spleen compartments. Ex vivo stimulation of CD4+ T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4+ T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.

Synergistic CRISPR/Cas9 Gene Editing and CAR T-cell Therapy for FLT3-Mutated in AML

Acute Myeloid Leukemia (AML) with FLT3 (FMS-like Tyrosine Kinase 3) mutations poses a significant challenge in oncology. This paper explores the potential of a combined treatment approach involving CRISPR/Cas9 gene editing and CAR T-cell therapy. CRISPR/Cas9 technology can precisely target and correct the FLT3 gene mutation responsible for AML proliferation. Simultaneously, CAR T-cell therapy harnesses the immune system to target FLT3-mutated AML cells using engineered T cells. While promising, these approaches come with challenges, such as off-target effects for CRISPR/Cas9 and complications like cytokine release syndrome for CAR T-cell therapy. Nevertheless, they offer a groundbreaking paradigm shift in AML treatment, potentially providing more effective and personalized therapies for patients with FLT3 mutations. Further research and clinical trials are essential to fully realize their potential and address associated hurdles. This combination therapy offers hope for improving outcomes in AML patients with FLT3 mutations, representing a significant advancement in precision oncology.

FLT3 Mutated Acute Myeloid Leukemia after CD19 CAR-t Cells.

Chimeric Antigen Receptor T-cells have improved the life expectancy of severely pretreated patients with aggressive hematological cancers; for this reason, therapy-related myeloid leukemias are becoming of great concern in this field, despite their clonal phylogenesis and mutational landscape have not been fully explored yet. This case discusses a 33-year-old man with refractory large B-cell lymphoma, treated with Chimeric Antigen Receptor T-cell (CAR-T) therapy as the 7th line of treatment. Despite a persistent partial response, the patient developed therapy-related acute myeloid leukemia (t-AML) six months post-CAR-T, revealing pre-existing clonal hematopoiesis. The myeloid malignancy exhibited an unusual hypocellular/dysplastic pattern, progressing to an established blast phase with cytopenia. Treatment with demethylating agents and BCL2 inhibitors proved ineffective, leading to t-AML with hyperleukocytosis and FLT3-ITD gain, resulting in the patient's death. This case underscores the impact of severe pretreatment and bone marrow impairment in CAR-T-associated t-AML, emphasizing their role over insertional mutagenesis. Furthermore, it highlights the retention of classic therapy-related leukemia characteristics, including the potential for acquiring FLT3 mutations and displaying dysplastic morphology in these secondary leukemias.

UBTF tandem duplications in pediatric myelodysplastic syndrome and acute myeloid leukemia: implications for clinical screening and diagnosis.

Recent genomic studies in adult and pediatric acute myeloid leukemia (AML) demonstrated recurrent in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF). These alterations, which account for approximately 4.3% of AML in childhood and about 3% in adult AML aged <60 years of age, are subtype-defining and associated with poor outcomes. Here, we provide a comprehensive investigation into the clinicopathological features of UBTF-TD myeloid neoplasms in childhood, including 89 unique pediatric AML and 6 myelodysplastic syndrome (MDS) cases harboring a tandem duplication in exon 13 of UBTF. We demonstrate that UBTF-TD myeloid tumors are associated with dysplastic features, low bone marrow blast infiltration, and low white blood cell count. Furthermore, using bulk and single-cell analyses, we confirm that UBTF-TD is an early and clonal event associated with a distinct transcriptional profile, whereas the acquisition of FLT3 or WT1 mutations is associated with more stem cell-like programs. Lastly, we report rare duplications within exon 9 of UBTF that phenocopy exon 13 duplications, expanding the spectrum of UBTF alterations in pediatric myeloid tumors. Collectively, we comprehensively characterize pediatric AML and MDS with UBTF-TD, and highlight key clinical and pathologic features that distinguish this new entity from other molecular subtypes of AML.