FLT3 Tyrosine Kinase Inhibitors Research Articles

Key role of glutamine metabolism in persistence of leukemic cells upon exposition to FLT3 tyrosine kinase inhibitors

Acute myeloid leukemias are a group of hematological malignancies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FMS-like tyrosine kinase 3 (FLT3) gene has led to the development of tyrosine kinase inhibitors such as quizartinib. However, achieving complete remission in patients remains challenging because these new tyrosine kinase inhibitors (TKIs) are unable to completely eradicate all leukemic cells. Residual leukemic cells persist during quizartinib treatment, leading to the rapid emergence of drug-resistant leukemia. Given that mitochondrial oxidative metabolism promotes the survival of leukemic cells after exposure to multiple anticancer drugs, we characterized the metabolism of leukemic cells that persisted during quizartinib treatment and developed metabolic strategies to eradicate them. In our study, employing biochemical and metabolomics approaches, we confirmed that the survival of leukemic cells treated with FLT3 inhibitors critically depends on maintaining mitochondrial metabolism, specifically through glutamine oxidation. We uncovered a synergistic interaction between the FLT3 inhibitor quizartinib and L-asparaginase, operating through antimetabolic mechanisms. Utilizing various models of persistent leukemia, we demonstrated that leukemic cells resistant to quizartinib are susceptible to L-asparaginase. This combined therapeutic strategy shows promise in reducing the development of resistance to FLT3 inhibitors, offering a potential strategy to enhance treatment outcomes.

Current knowledge about FLT3 gene mutations, exploring the isoforms, and protein importance in AML.

Acute myeloid leukaemia (AML) is a complex haematological malignancy characterised by diverse genetic alterations leading to abnormal proliferation of myeloid precursor cells. One of the most significant genetic alterations in AML involves mutations in the FLT3 gene, which plays a critical role in haematopoiesis and haematopoietic homeostasis. This review explores the current understanding of FLT3 gene mutations and isoforms and the importance of the FLT3 protein in AML. FLT3 mutations, including internal tandem duplications (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD), occur in 25-30% in AML and are associated with poor prognosis. FLT3-ITD mutations lead to constitutive activation of the FLT3 signalling pathway, promoting cell survival and proliferation. FLT3-TKD mutations affect the tyrosine kinase domain and affect AML prognosis in various ways. Furthermore, FLT3 isoforms, including shorter variants, contribute to the complexity of FLT3 biology. Additionally, nonpathological polymorphisms in FLT3 are being explored for their potential impact on AML prognosis and treatment response. This review also discusses the development of molecular treatments targeting FLT3, including first-generation and next-generation tyrosine kinase inhibitors, highlighting the challenges of resistance that often arise during therapy. The final chapter describes FLT3 protein domain rearrangements and their relevance to AML pathogenesis.

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.

Гилтеритиниб — новая возможность в лечении рецидивов и рефрактерных острых миелоидных лейкозов с мутацией в гене FLT3: обзор литературы и описание трех собственных клинических наблюдений

Acute myeloid leukemias (AML) are the most ubiquitous of all adult leukemias. The prognosis of the disease depends on its genetic profile. The mutation in FLT3 gene, which codes FMS-like tyrosine kinase 3, is observed in 1/3 of patients and is responsible for a high rate of relapses. The prognosis of relapsed/refractory FLT3-positive AML is extremely poor. The standard intensive therapy rarely yields long-term responses. The new first- and second-generation FLT3 tyrosine kinase inhibitors enriched treatment opportunities for patients with this mutation. Gilteritinib, a potent second-generation FLT3-ITD/TKD inhibitor, is a new effective and well tolerated drug for the treatment of relapsed/refractory FLT3-positive AML. Due to its efficacy, low toxicity, and good manageability, this drug can be administered to all patients, including the elderly or those with severe comorbidities and complications of previous therapy. Besides, this drug can be used in outpatient units. The present paper contains three case reports dealing with different clinical situations in patients with FLT3-positive AML treated with gilteritinib in real-world clinical practice.

Abstract C148: Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation

Abstract Introduction: Fusion oncoproteins involving the ROS1 receptor tyrosine kinase have been established as validated therapeutic targets in non-small cell lung cancer. FDA-approved ROS1 tyrosine kinase inhibitors (TKIs) such as crizotinib and entrectinib have shown substantial response rates in these patients, leading to improved outcomes. Historically, the solvent-front ROS1 kinase mutation, G2032R, was a recurrent resistance liability to first generation ROS1 TKI. Next-generation TKIs, including NVL-520, repotrectinib, and taletrectinib are active against the ROS1 G2032R and shown activity in both treatment-naïve and ROS1 G2032R acquired resistance setting. We and others showed that ROS1 L2086F a mutation in catalytic-spine 6 within the kinase domain confers resistance to all first and next-generation type I binding mode TKIs. Cabozantinib, a type II ROS1 inhibitor, is the only FDA-approved TKI that has showed clinical activity against ROS1 L2086F. Recent reports indicated that gilteritinib, an FLT3 TKI, exhibits activity against ALK L1256 (homologous to ROS1 L2086F). In this study, we investigated the efficacy of gilteritinib compared to cabozantinib in ROS1 L2086F resistance model systems. Methods: We tested the effectiveness of ROS1 TKI compared to gilteritinib in Ba/F3 cell lines expressing CD74-ROS1 and EZR-ROS1 resistant mutations. Additionally, we quantified the efficacy of cabozantinib and gilteritinib in ROS1 L2086F using soft agar assays. We engineered CRISPR-Cas9 genome-edited CUTO-28 cells expressing endogenous L2086F mutation, and to validate the activity of gilteritinib and cabozantinib against ROS1 L2086F. Results: The ROS1 kinase mutations F2004C, G2032R and L2086F are resistant to entrectinib inhibition. Cabozantinib, consistent with our previous published data, retains inhibitory activity against ROS1 G2032R and ROS1 L2086F, however the ROS1 F2004C mutation imposes cabozantinib-resistance. Gilteritinib, intriguingly, is active with IC50 ≤ 25 nM against ROS1 F2004C and ROS1 L2086F but not ROS1 G2032R. Immunoblotting studies showed on-target inhibition of ROS1 auto-phosphorylation and effector ERK1/2 activation in the case of ROS1 L2086F mutant cells treated with cabozantinib and gilteritinib. Conclusion: Gilteritinib demonstrates inhibitory activity against ROS1 L2086F mutation. Currently, patients with ROS1 L2086F resistance can be treated with cabozantinib as a second-line treatment option. The preclinical data presented herein suggest that gilteritinib could be an additional, potentially viable alternative to target the highly resistant ROS1 L2086F mutation. Additional studies are needed to explore the use of gilteritinib for ROS1 rearranged NSCLC. Citation Format: Rajat Thawani, Clare Keddy, Matteo Repetto, Kristen Jones, Katelyn Nicholson, Zoe Beach, Alexander Drilon, Monika Davare. Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C148.

Mediator Kinase/CDK8 Inhibition As a Strategy to Improve FLT3 Inhibitor Activity in Acute Myeloid Leukemia

Acquired resistance to targeted FLT3 tyrosine kinase inhibitors (TKIs) remains a major barrier to durable clinical responses for patients with FLT3-mutant AML. Multiple resistance mechanisms to next generation FLT3 TKIs converge upon RAS/MAPK pathway activation, including protective cytokine stimuli of the bone marrow microenvironment or through secondary oncogenic RAS mutations. RAS mutations also decrease efficacy of the BCL2 inhibitor venetoclax now widely used in AML, suggesting an anti-apoptotic influence of MAPK signaling. These findings highlight a significant unmet need for novel therapeutic combinations to address MAPK-mediated resistance in AML. We report results from a genome-wide CRISPR interference (CRISPRi) apoptosis screen using FLT3-ITD mutant MOLM-14 cells treated with the FLT3 TKI gilteritinib while cultured in HS5 conditioned media (CM) to mimic cell-extrinsic MAPK-driven TKI resistance. We identified RNA pol II and Mediator kinase/CDK8 ascritical nodes for AML cell survival in the setting of FLT3 inhibition. We further demonstrated that CDK8 inhibition with the small-molecule SEL120 re-sensitized multiple FLT3-mutant AML cell lines and primary samples to gilteritinib. We also performed a second CRISPRi apoptosis screen to identify hits that sensitize to gilteritinib in a model of cell-intrinsic MAPK-mediated resistance using MOLM-14 cells that harbor secondary NRAS-G12C mutation.We used Enrichr to perform an integrated analysis and validated the Core Mediator Complex and CDK8 knockdown genes as cellular components significantly enriched among sensitizing hits in both screens. The Mediator subunit MED12 was identified as a top gene level hit that strongly sensitized cells to gilteritinib in both screens, further emphasizing that Mediator kinase and its transcriptional regulation may be a central pathway engaged in MAPK-dependent survival in FLT3-mutant AML. To better understand potential mechanisms of increased combinatorial CDK8i/FLT3i activity, we performed RNA-seq in MOLM-14 cells treated with gilteritinib, SEL120, or the combination in HS5 CM. Using GSEA we found that gilteritinib stimulated adaptive interferon/inflammatory gene signatures at 16h drug treatment, but this response was restrained by addition of SEL120. Additionally, we identified a striking upregulation of the lineage-controlling transcription factor IRF8 and its gene expression signature with concomitant silencing of SPI1 transcriptional output in cells treated with gilteritinib/SEL120. CDK8i treatment alone in AML has previously been shown to increase expression of super-enhancer (SE) associated genes, including IRF8. Interestingly, SPI1was also a significant gene-level hit in both CRISPRi screens. Using CRISPRi we repressed IRF8 expression in MOLM-14, MOLM-14 NRAS-G12C, and MV411 (another FLT3-mutant line) cells and found this significantly blunted the combinatorial pro-apoptotic effect of SEL120, implying that induction of IRF8 expression plays a significant role in driving response to combined gilteritinib/SEL120. We finally assessed in vivo activity of gilteritinib combined with SEL120. We first performed pilot toxicity studies in NSG mice and determined that gilteritinib 30 mg/kg, SEL120 30 mg/kg, or the combination dosed 5 days/week by oral gavage (OG) were regimens well tolerated by mice. We then engrafted luciferase-tagged MOLM-14 and MOLM-14 NRAS-G12C cells into NSG mice and treated for 4-weeks with gilteritinib, SEL120, or the combination. We found gilteritinib/SEL120 extended survival in both MOLM-14 and MOLM-14 NRAS-G12C models. Similar efficacy studies are ongoing in FLT3-mutant patient-derived xenograft (PDX) models with NSGS mice and will be reported. Our results indicate that FLT3-mutant AML cells employ an adaptive inflammatory response to evade FLT3i-induced apoptosis, and CDK8i represses this adaptation while concomitantly increasing the ratio of IRF8 to SPI1-driven gene expression programs. We speculate this latter effect may in part alter an AML cell's differentiation state to sensitize cells to apoptosis, though further study to explore this hypothesis is needed. Our in vitro and in vivo efficacy data further validated combined FLT3i/CDK8i as a promising investigational strategy to pre-empt or overcome MAPK-mediated FLT3 TKI resistance.

Epitope Edited Hematopoietic Stem Cells to Enable Synergistic Immunotherapy Combinations for Acute Myeloid Leukemia

Despite treatment advances, acute myeloid leukemia (AML) is still associated with an unfavorable outcome for >50% of patients. Whereas novel immunotherapies, such as CAR-T cells, bispecific and toxin conjugated antibodies (mAb), demonstrated clinical efficacy when targeting dispensable lineage antigens (Ag), such as CD19 in B-ALL, the same approach cannot be exploited for AML, due to lack of actionable leukemia-restricted Ags. Suitable targets are shared with healthy progenitor or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. Several AML immunotherapies are currently under development, but their use is restricted to a limited time window, likely insufficient for disease eradication. To address these issues, we reasoned that precise modification of the targeted epitopes in donor HSPC used in HSCT results in loss of mAb recognition, without affecting normal protein expression, regulation, and function ( Nature, accepted). Epitope editing allows targeting genes essential for leukemia survival regardless of shared expression or functional role in normal HSPC, thus minimizing the risk of tumor immune escape by Ag loss or downregulation. Cytokine receptors such as FLT3, KIT and CD123 are found in >85% of AML cases and their mutation (e.g., FLT3-ITD) or overexpression is associated with poor prognosis. We identified amino acid substitutions in the FLT3, KIT and CD123 extracellular domains that preserve physiologic surface expression, ligand-binding, kinase phosphorylation, colony-forming capacity, proliferative response, transcriptional and phospho-proteomic profile of CD34+ HSPCs but avoid detection by a therapeutic monoclonal Ab. Cells expressing these variants were resistant to CAR-T killing and did not induce CAR activation and proliferation during in vitro co-culture. We were able to introduce these mutations into CD34+ HSPCs by adenine base editing (ABE) with high efficiencies (90%, 85% and 75% for FLT3, KIT and CD123, respectively) without the need for dsDNA breaks. After xenotransplant into NBSGW mice, FLT3, KIT or CD123 epitope-editedHSPC sustained long-term multilineage hematopoiesis indicating editing and preserved functionality of HSCs. Upon treatment with FLT3 CAR-T in vivo, we observed sparing of human CD34+38- HSPCs, granulo-mono progenitors (GMP), B-cells and B-progenitors in the bone marrow of mice engrafted with FLT3-edited HSPCs compared to AAVS1 controls. Treatment with CD123 CAR-T cells showed protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. We next generated advanced in vivo models with co-engraftment of human HSPCs and patient-derived AML xenografts (PDX), to demonstrate the selective resistance of epitope edited HSPCs and their progeny, while PDX were eradicated by FLT3 or CD123 CAR-T. To further enhance therapeutic efficacy, we are now exploring the combination of epitope-edited HSPCs and pharmacological agents with potential synergistic effects with CAR-T cells. FLT3 tyrosine kinase inhibitors (e.g., Crenolanib) have the potential to enforce surface expression of FLT3 by impairing its intracellular recycling, thus enhancing CAR-T mediated killing, but their use may be limited by overlapping toxicities on healthy hematopoiesis, in particular in the post-HSCT setting. To assess if epitope-editing could prevent not only CAR- but also TKI-mediated toxicities, we treated mice co-engrafted with FLT3- or AAVS1- edited HSPCs and a FLT3-ITD+ AML PDX with combinations of FLT3-CAR and a 2-week course of Crenolanib. Leukemia burden was reduced by Crenolanib alone and eradicated by FLT3-CAR-T. Critically, we observed preservation of healthy hematopoietic lineages only in mice engrafted with epitope-edited HSPCs, including the CAR+Crenolanib group, while AAVS1 controls showed overlapping off-tumor toxicity when treated with the CAR+Crenolanib combination. In conclusion, we believe that epitope edited HSPCs may not only enable safe and effective CAR-T immunotherapies for AML, but also allow their combination with pharmacological blockade of leukemia survival/proliferative pathways to achieve synthetic lethality mechanisms, while still avoiding dose-limiting toxicities. Further exploration of immunotherapy-synergistic combinations will be fundamental to improve the outcomes of difficult-to-treat high-risk AML patients.

Clinico-Genomic Characterization of AML Patients Based on IL2RA (CD25) Expression Uncovers an Association with Stem Cell Signatures and FLT3-ITD Status and Informs Drug Combinations

Background Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes, thus there remains a need to integrate molecular information to identify patients most likely to respond to drugs and combinations. Disease segmentation based on transcriptomics has provided valuable insight into disease risk and the likelihood of response to targeted compounds. IL2RA(CD25) is a receptor expressed on both AML leukemic and immune cells, and has shown initial promise as a potential dual target of leukemic blasts and regulatory T (Treg) cells in pre-clinical models (Pousse et al. Front. Oncol. 2023). We used a systems approach based on a recently described transcriptomic classifier for AML (Hamidi et al. ASH 2021) and other molecular tools to characterize the association between IL2RA expression levels and AML genomic markers, clinical features and patient outcomes. In addition, we evaluated IL2RA in relation to ex vivo drug sensitivity, to identify patient segments who are most likely to benefit from CD25-targeting drugs. Methods BEAT-AML (NCT01728402) RNAseq data from patient samples, associated ex vivo drug sensitivity data (N=283), was VOOM normalized. Patients were binned into quartiles based on IL2RA expression. Gene signature scoring was performed using xCell cell type enrichment algorithm and GSVA for Hallmark pathways and scRNAseq signature based on Van Galen et al. Cell 2019. Associations were performed with clinical outcome (log-rank test), gene signatures (Spearman correlation), mutations (Wilcoxon test) and ex vivo drug sensitivity (Kruskal Wallis test). Results We characterized the BeatAML dataset and found that elevated IL2RA expression associates with inferior overall survival (p=0.014) and high-risk features. In addition, we identified a strong association between IL2RA expression and FLT3-ITD status (Figure), as well as other genetic alterations. Using correlation analyses, we established an association between IL2RA expression levels and “primitive” AML signatures (leukemic stem cell (LSC), R=0.4, p=6.8x10-12, hematopoietic stem cell (HSC)-like, R=0.43, p=6.8x10-14) and Tregs (R=0.47, p=1.7x10-15), and an anti-correlation with promonocytic signatures (R=-0.23, p=1.1x10-4). Interestingly, HSC-like and Treg signatures were also correlated (R=0.19, p=0.00087), consistent with an association between stem cell abundance and a repressive immune microenvironment. Finally, using a multivariate model adjusting for the effect of Tregs and LSCs, the prognostic value of IL2RA remained significant (p=0.029). We previously used unsupervised machine learning clustering based on consensus non-negative factorization (cNMF) to discover novel transcription-based classification (Hamidi ASH 2021). Using this methodology, we identified a strong correlation between IL2RA expression in cNMF subtypes (p=1.6x10-10). Moreover, this method uncovered patient subtypes in which IL2RA correlates with LSC (cNMF 6.3), Treg (6.4, 6.6) or both signatures (6.1, 6.2). This differential association was independent of prognostic category, maturation state or venetoclax sensitivity (Table). Transcription-based classifiers have been shown to be highly predictive of ex vivo drug sensitivity. Consistent with an association between IL2RA expression and FLT3-ITD status, we identified strong correlations between IL2RA and sensitivity to FLT-3 and other tyrosine kinase inhibitors, providing a rationale for combining these compounds with CD25-targeting therapeutics. In contrast, there was no correlation between IL2RA levels and venetoclax AUC (p=0.25), while the cNMF classification system similarly revealed that patient segments with increased IL2RA expression had heterogeneous venetoclax sensitivity (Table), supporting CD25 targeting in patients who may not respond to venetoclax. Conclusion AML patients with elevated IL2RA expression have inferior prognosis, enrichment of stem-like and Treg signatures and FLT3-ITD alterations. Transcriptomics-based clustering could be used to guide combination therapies based on potential impact of CD25 targeting on Tregs, leukemic cells, or both populations. Ex vivo drug sensitivity analyses support combinations of CD25-targeting agents with FLT3 inhibitors, as well as venetoclax. Additional work to evaluate these and other combinations using functional assays is warranted.

Metformin Potentiates Gilteritinib Sensitivity Via Targeting PLK1 Signaling: A Strategy to Improve Outcomes and Reduce Costs in Treating FLT3-Mutated Acute Myeloid Leukemia

FLT3 mutations (including FLT3-ITD and FLT3-TKD), occurring in ~30% of acute myeloid leukemia (AML) patients, signal poor prognosis due to elevated relapse risk. Albeit FLT3 tyrosine kinase inhibitors (FLT3 TKI) demonstrated efficacy in treating FLT3-mutated AML in the clinic, there are issues such as high cost, side effects, and high rate of drug resistance/relapse. Hence, an affordable, efficient treatment with lower side effects is needed. Metformin (MET), a cost-effective anti-diabetic drug with minimal side effects, has demonstrated anti-tumor effects (Dowling R et al. Cancer Res. 2007). Here we aimed to determine if combining MET with FLT3 TKI, like Gilteritinib (GLT), could enhance efficacy, reduce side effects and costs in treating FLT3-mutated AML. To this end, we first assessed the IC50 values of MET and GLT across various AML cell lines including those carrying FLT3/ITD (MOLM13, MV4-11, and MA9.3-ITD), FLT3 TKI resistant cells (MOLM13-RES) and those carrying FLT3 wild-type (FLT3/WT) (THP1, NOMO1 and Kasumi-1). MET's IC50 S were consistent (7.284 -12.76 mM) (Fig. 1A), while GLT's varied, with lower values in FLT3/ITD cells (10 nM) and higher in FLT3 TKI resistant (241 nM) and FLT3/WT cells (334-1,053 nM) (Fig. 1B). Using the Chou-Talalay Method, a Combination Index (CI) below 1 in FLT3/ITD and MOLM13-RES cells indicated synergy, whereas it showed antagonism (CI>1) in FLT3/WT cells (Fig. 1C). Both MET and GLT induced cell cycle arrest and apoptosis, with Combo1 (GLT 5 nM + MET 5 mM) matching GLT 10 nM effects; Combo2 (GLT 10 nM + MET 10 mM) amplified these effects (Fig. 1D and E). We next evaluated the potential synergistic effect of MET and GLT in treating TKI-resistance AML in vivo using NRGS mice xeno-transplanted with MOLM13-RES cells. Both bioluminescence imaging and flow cytometry assays showed each treatment alone reduced leukemia burden, and their combination had a synergistic effect, reducing AML burden dramatically (Fig. 1F). While MET or GLT alone doubled overall survival compared to vehicle treatment (56-62 days vs. 26 days), ~90% of the mice treated with the Combo survived over 120 days (Fig. 1G) and their leukemia burden was still minimal, highlighting the synergistic effect and the promising therapeutic potential of this combination in treating FLT3-mutated AML (even with TKI-resistance). To understand the underlying molecular mechanism(s), we performed RNA-seq with vehicle- or drug-treated MOLM13-RES cells, followed by pathway analysis and validation/mechanistic studies, we identified Polo-like kinase 1 (PLK1) as a key target of MET and GLT (Fig. 1H). PLK1's expression is notably suppressed by both treatments (Fig. 1I). Mimicking MET treatment, PLK1 knockdown enhanced GLT sensitivity, reducing IC50 from 259.2 nM to 47-60 nM (Fig. 1J). Conversely, PLK1 overexpression increased GLT's IC50 7-fold (13.07 nM to 95.94 nM) (Fig. 1K), largely reversing MET's effect (data not shown). PLK1, a crucial serine/threonine-protein kinase, influences multiple signaling pathways such as MAPK and mTOR pathways. Upon treating MOLM13 or MOLM13-RES cells with MET and GLT alone or combined, the PLK1 levels and phosphorylation levels of FLT3, STAT5, ERK, and mTOR were decreased, with the most significant decrease occurred upon the combined treatment (Fig. 1L). Depletion and overexpression of PLK1 decreased and increased these phosphorylation levels, respectively (data not shown). Together, our findings suggest that the synergistic therapeutic effect of MET and GLT in treating FLT3-mutated AML is likely through synergistically targeting PLK1 and its downstream pathways. Meanwhile, we executed a retrospective clinical study of FLT3/ITD AML patients who were also diagnosed with diabetes. Metformin users showed a notably longer median OS (13.2 months) than non-MET users (4.5 months, P=0.0197) (Fig. 1M). Despite FLT3/ITD status, FLT3 TKIs were seldom used in China due to high cost. Additionally, PLK1 level was dramatically higher in FLT3/ITD AML cases than in both healthy and FLT3/WT cases (P<0.001) (Fig. 1N), implying PLK1's role in FLT3/ITD's poor prognosis. In summary, our findings suggest a potential synergistic approach in treating FLT3-mutated AML using MET and GLT. This approach could reduce side effects and costs (crucial for developing countries) and improve outcomes. Larger clinical trials are needed to confirm these promising initial findings.

Gilteritinib and Venetoclax for Relapsed/Refractory FLT3-Mutated Acute Myeloid Leukemia - a Real-World Multicenter Retrospective Study

Introduction: Relapsed/Refractory (R/R) FLT3-mutated AML is a challenging clinical scenario with a dismal outcome. Gilteritinib, a selective and potent oral FLT3 tyrosine kinase inhibitor (TKI), is approved for treatment of FLT3 mutated R/R AML, and was shown to improve response rates and overall survival (OS) as compared to standard chemotherapy (Perl et al, NEJM 2019). However, remission rates and long-term survival with gilteritinib monotherapy are still unsatisfactory, highlighting the need for new therapies to improve patient outcomes. Recently, the combination of gilteritinib and venetoclax (Gilt-Ven) was shown to achieve a response rate of 75% in R/R FLT3 AML patients (Daver et al, JCO 2022). Data regarding Gilt-Ven based combinations in patients outside of clinical trials is lacking. We therefore aimed to investigate the characteristics and outcomes of real-world patients with R/R FLT-3 mutated AML treated with Gilt-Ven based combinations. Methods: A multicenter retrospective cohort study in seven academic centers in Israel. Demographic, clinical and treatment related data were collected. The decision to treat a patient with Gilt-Ven (with or without azacitidine) as well as dosing were at discretion of the treating physician. Results: Eighteen patients were treated with Gilt-Ven based therapies for FLT-3 mutated R/R AML between January 2018 and June 2023. Median follow-up time was 5 months (range 0-24). Median age at treatment initiation was 48 years (range 24-79). Twelve patients (66.7%) had relapsed disease, while 6 (33.3%) had refractory disease. Thirteen patients (72.2%) had a normal karyotype; two had complex karyotype and one had monosomal karyotype. Sixty-one percent had prior therapy with a FLT3 inhibitor, and 33% had prior venetoclax exposure. Eighty-eight percent received a prior intensive induction regimen and 61% had prior Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT) (Figure 1A). Most patients received Gilt-Ven doublet, while 27% received Gilt-Ven in combination with other agents (three patients with azacitidine, one with low-dose cytarabine and one with FLAG-IDA). The median dose for gilteritinib was 120 (range, 80-120) mg and all patients received 400 mg venetoclax (or equivalent dose adjustment to azoles). Overall Response Rate (CR+CRi+MLFS) was 83%; Complete remission rate was 50% (Figure 1B). The median number of cycles was 1.5 (range 1-34) for the whole cohort. Five patients (27%) proceeded to alloHSCT. Of these, four patients required one course of Gilt-Ven, and one patient received 6 courses and proceeded to AlloHSCT after a subsequent relapse and another line of therapy. Median OS was not reached for all patients (range 6-NR), but was only 6 months (range 0-10) for patients who did not proceed to AlloHSCT. Hematologic adverse events were most common with 89% of patients experiencing grade 3/4 neutropenia; however, infections were relatively uncommon with only 17% of patients experiencing grade 3/4 Infection. Transaminitis grade 1/2 was observed in 38.9% of patients while no patient developed grade 3/4. No cases of differentiation syndrome were recorded. Conclusion: In a real-world setting, Gilt-Ven based therapy is effective for heavily pre-treated patients with FLT-3 mutated R/R AML, especially as a bridge to AlloHSCT.

PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20–30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Healthcare resource utilization and costs during first salvage therapy for relapsed or refractory acute myeloid leukemia in the United States

Real-world US healthcare resource utilization (HRU) and costs during first salvage therapy for relapsed/refractory (R/R) acute myeloid leukemia (AML) are described using IBM MarketScan® data (1/1/2007–6/30/2020). Treatments included high- (HIC) and low-intensity chemotherapy (LIC) alone, and gilteritinib, other FLT3 tyrosine kinase inhibitors (TKIs), and venetoclax with or without chemotherapy. Patients were diagnosed with R/R AML at ≥18 years of age between 1/1/2017–12/31/2019. Patient monthly all-cause HRU and costs were analyzed using a fixed-effects model. Data from 399 patients were analyzed (HIC, n = 104; LIC, n = 133; gilteritinib, n = 14; other FLT3 TKIs, n = 68; venetoclax, n = 80). Inpatient HRU was generally highest with HIC, whereas outpatient HRU was generally highest with LIC and venetoclax. Total all-cause incremental monthly costs appeared to be highest with HIC ($171,982) and similar for LIC ($60,512), gilteritinib ($47,218), other FLT3 TKIs ($43,218), and venetoclax ($77,566). Results highlight HRU and cost differences for R/R AML during first salvage therapy.

Gilteritinib plus azacitidine and venetoclax for FLT3-ITD mutated relapsed/refractory acute myeloid leukemia.

e19024 Background: Gilteritinib is a second-generation FLT3 tyrosine kinase inhibitor that has been shown significantly increase the survival of relapsed/refractory (r/r) FLT3-mutated acute myeloid leukemia (AML) patients. We investigated the clinical benefit of gilteritinib plus azacitidine and venetoclax in the treatment of r/r FLT3-ITD mutated AML in the real word. Methods: Gilteritinib was given by oral administration at a dose of 80 or 120mg/d for each cycle. Azacitidine was intravenously administrated at a dose of 75 mg/m2/day d1-7. Venetoclax was given by oral administration at a dose of 100mg/d then ramp-up at 400mg/d, some patients were administrated at a dose of 400mg/d due to prior exposure. The composite complete response (CRc) rate (complete response [CR] and CR with incomplete blood count recovery [CRi]) were assessed, and then the overall survival (OS), event-free survival (EFS), relapse-free survival (RFS) was evaluated. Results: We retrospectively analyzed the outcomes of gilteritinib plus azacitidine and venetoclax in 14 r/r FLT3-ITD mutated AML patients. The CRc rate of the triple therapy was 57.1% (CR, n = 5, 35.7%; CRi, n = 3, 21.4%), in which, all patients achieved minimal residual disease (MRD) negative measured by multi-color flow cytometry. At a median follow-up of 136.5 days, the median OS was 216.0 days, the median EFS was 97.0 days, and the median RFS were 184 days. The common grade 3/4 hematologic adverse events included neutropenia (n = 14, 100.0%), anemia (n = 12, 85.7%), and thrombocytopenia (n = 11, 78.6%). The common grade 3/4 non-hematologic adverse events were pulmonary infection (n = 5, 35.7%), other adverse events were relatively less and milder. Conclusions: In conclusion, the current data demonstrated promising response of the triple therapy in r/r FLT3-ITD mutated AML patients, and the regimen represented a potential option as a bridge to transplant. Prospective studies are warranted to further define the risk or benefit for more patients.

Loss of MiR-155 Sensitizes FLT3-ITD+AML to Chemotherapy and FLT3 Inhibitors via Glycolysis Blocking by Targeting PIK3R1.

FLT3 tyrosine kinase inhibitors in combination with chemotherapy have shown some success in patients with FLT3 mutations. But a variety of mechanisms have led to the rapid resistance to the treatment. One of the most prominent is the metabolic alteration on aerobic glycolysis. We aim to explore the role of a high expressing microRNA, miR-155, in mediating resistance to chemotherapy and FLT3 inhibitor treatment. The deep sequencing data mining revealed the connection between glycolysis and drug resistance. MV411 cells with miR-155 knockout (KO) not only had increased sensitivity to FLT3 inhibitors but also Adriamycin (ADM) treatment. When combined with glycolysis inhibition the treatment response in MV411 cells further increased. Whereas in miR-155 KO cells, a lower glucose consumption level and lactic acid level were observed, and western blotting showed a decreased expression of key enzymes in glycolysis pathways. A negative correlation between PIK3R1 and miR-155 level can be observed in the sequencing data from FLT3-ITD+ AML patients. Moreover, luciferase reporter assay revealed that the 3'UTR of PIK3R1 mRNA can interact with the seed sequence of miR-155-5p. In conclusion, the loss of miR-155 increased treatment sensitivity to both chemotherapy and FLT3 inhibitors in FLT3-ITD+ AML cells via glycolysis blocking by targeting PIK3R1.

Microenvironmental CXCL12 deletion enhances Flt3-ITD acute myeloid leukemia stem cell response to therapy by reducing p38 MAPK signaling.

Fms-like tyrosine kinase 3 (Flt3) tyrosine kinase inhibitors (Flt3-TKI) have improved outcomes for patients with Flt3-mutated acute myeloid leukemia (AML) but are limited by resistance and relapse, indicating persistence of leukemia stem cells (LSC). Here utilizing a Flt3-internal tandem duplication (Flt3-ITD) and Tet2-deleted AML genetic mouse model we determined that FLT3-ITD AML LSC were enriched within the primitive ST-HSC population. FLT3-ITD LSC showed increased expression of the CXCL12 receptor CXCR4. CXCL12-abundant reticular (CAR) cells were increased in Flt3-ITD AML marrow. CXCL12 deletion from the microenvironment enhanced targeting of AML cells by Flt3-TKI plus chemotherapy treatment, including enhanced LSC targeting. Both treatment and CXCL12 deletion partially reduced p38 mitogen-activated protein kinase (p38) signaling in AML cells and further reduction was seen after treatment in CXCL12 deleted mice. p38 inhibition reduced CXCL12-dependent and -independent maintenance of both murine and human Flt3-ITD AML LSC by MSC and enhanced their sensitivity to treatment. p38 inhibition in combination with chemotherapy plus TKI treatment leads to greater depletion of Flt3-ITD AML LSC compared with CXCL12 deletion. Our studies support roles for CXCL12 and p38 signaling in microenvironmental protection of AML LSC and provide a rationale for inhibiting p38 signaling to enhance Flt3-ITD AML targeting.

A Phase I/II Study of Combination of ASTX727, Gilteritinib and Venetoclax in Patients with Relapsed/Refractory FLT3 Mutated Acute Myeloid Leukemia (AML) and Frontline FLT3 Mutated AML Patients Unfit for Chemotherapy

Background:FLT3 gene mutations occur in approximately 30% of patients (pts) with AML and are associated with worse survival and higher relapse risk. Gilteritinib, a selective potent oral FLT3 tyrosine kinase inhibitor (FLT3i), improves survival in pts with relapsed FLT3 mutated AML. ASTX727 is an oral fixed-dose combination of decitabine (35 mg) and cedazuridine (a cytidine deaminase inhibitor; 100 mg) approved for treating pts with myelodysplastic syndrome (MDS). We are evaluating the safety and efficacy of the combination of ASTX727, gilteritinib, and venetoclax (VEN) in pts with relapsed or refractory FLT3-mutated AML and older/unfit pts unable to receive chemotherapy. Methods: In the phase I, pts with R/R FLT3-mutated AML or high-risk (HR) MDS or chronic myelomonocytic leukemia (CMML) are eligible. FLT3-ITD and/or FLT3-TKD mutations were allowed. In the phase II, the study will also enroll newly diagnosed pts with FLT3-mutated AML ineligible for induction therapy. Pts are required to have an ECOG performance status ≤3, total bilirubin ≤2.5 x upper limit of normal (ULN), ALT/AST ≤3 x ULN, and creatinine clearance ≥30 mL/min. Hydroxyurea or 1 dose of cytarabine up to 1000 mg was allowed in pts with proliferative disease to lower white blood count to ≤ 25 x 109/L before initiation of study therapy. In the phase 1, we evaluated two dose levels of gilteritinib (80 mg and 120 mg daily) continuously with fixed dosages of ASTX727 (35/100 mg daily on days 1-5) and VEN (400 mg daily, dose adjusted for concomitant azoles, for up to 28 days). Cycles are repeated every 28 days for up to 24 cycles, followed by indefinite therapy with gilteritinib as maintenance. During cycle 1, VEN was given at a dose of 100 mg on day 1, 200 mg on day 2, and 400 mg daily onwards. Bone marrow (BM) aspiration was performed on day 21, and VEN was held in pts with BM blasts <5% or aplasia. During cycles 2-24, VEN was given at 400 mg daily (dose adjusted for concomitant azoles) on days 1-21 but dose reductions depending on tolerance were permitted. Gilteritinib continued at the enrolled dose in cycles 2-24. Results: Between 11/2021 and 6/2022, 8 patients (7 AML and 1 HR-CMML) have been enrolled in the phase 1 portion of the trial (Table 1). Their median age was 57 years (range, 38-83). Median number of prior lines of therapy was 1 (range, 1-10). Four pts (50%) had a previous allogeneic hematopoietic stem cell transplant (HSCT), 6 pts (75%) had prior treatment with hypomethylating agent (HMA)+VEN, and 3 pts (27%) received prior FLT3i. None received prior gilteritinib. Cytogenetics was diploid in 4 pts, complex in 3 pts, and miscellaneous in 1 pt. The median FLT3-ITD allelic ratio (AR) was 0.4 (range, 0.11-10.60), with 50% having a high AR (≥0.5). All pts had a FLT3-ITD mutation and no pt had a FLT3-TKD mutation. NPM1 co-mutation was present in 4 pts (50%), followed by DNMT3A (37%) and IDH2 (37%). Six pts have been enrolled on dose level 1 (gilteritinib 80 mg) and 2 pt on dose level 2 (gilteritinib 120 mg). No DLTs have been observed in either dose level. Myelosuppression was frequent, requiring dose adjustment of ASTX727 and/or VEN in cycle 2 onward in 5 pts. Grade 3 non-hematologic adverse events included: sepsis (25%), bacteremia (25%), pneumonia (13%), and other infection (25%). No tumor lysis syndrome, differentiation syndrome, or QTc prolongation was observed. The 30-day and 60-day mortality rates were 0% and 13%, respectively. The overall response rate (ORR) was 87% with 4 (50%) pts achieving CR/CRi (1 CR, 3 CRi) and 3 (37%) pts achieving MLFS. One pt withdrew from the study in cycle 1 and was not evaluable for response. The ORR in pts with prior HMA+VEN was 83% (3 CRi, 2 MLFS). Five of 7 responders (71%) achieved the best response within 1 cycle and 2 (29%) within 2 cycles. Pts received a median of 2 cycles of therapy (range, 1-6). With a median follow-up of 6 months, median overall survival was not reached (range, 1.2-8+ months), and the estimated 6-month overall survival is 70%. One responding pt proceeded to HSCT. Two pts have died, 1 in CRi due to an unknown cause, and 1 due to disease progression and sepsis. Conclusion: The combination therapy with ASTX727, VEN, and gilteritinib appears to be effective and safe in R/R FLT3 mutated AML and HR-MDS. Myelosuppression was frequent but manageable. Continued accrual is anticipated to define the optimal triplet regimen in both R/R and newly diagnosed FLT3-mutated AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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

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

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.

Protein tyrosine kinase 2b inhibition reverts niche-associated resistance to tyrosine kinase inhibitors in AML

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

Gilteritinib monotherapy for relapsed/refractory FLT3 mutated acute myeloid leukemia: a real-world, multi-center, matched analysis.

Patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Gilteritinib is a FLT3 tyrosine kinase inhibitor (TKI) recently approved for patients with R/R AML. We aimed to characterize real-world data regarding gilteritinib treatment in FLT3-mutated R/R AML and to compare outcomes with matched FLT3-mutated R/R AML patients treated with chemotherapy-based salvage regimens. Twenty-five patients from six academic centers were treated with gilteritinib for FLT3-mutated R/R AML. Eighty percent were treated with a prior intensive induction regimen and 40% of them received prior TKI therapy. Twelve patients (48%) achieved complete response (CR) with gilteritinib. The estimated median overall survival (OS) of the entire cohort was eight (CI 95% 0-16.2) months and was significantly higher in patients who achieved CR compared to those who did not (16.3months, CI 95% 0-36.2 vs. 2.6months, CI 95% 1.47-3.7; p value = 0.046). In a multivariate cox regression analysis, achievement of CR was the only predictor for longer OS (HR 0.33 95% CI 0.11-0.97, p = 0.044). Prior TKI exposure did not affect OS but was associated with better event-free survival (HR 0.15 95% CI 0.03-0.71, p = 0.016). An age and ELN-risk matched comparison between patients treated with gilteritinib and intensive salvage revealed similar response rates (50% in both groups); median OS was 9.6months (CI 95% 2.3-16.8) vs. 7months (CI 95% 5.1-8.9) in gilteritinib and matched controls, respectively (p = 0.869). In conclusion, in the real-world setting, gilteritinib is effective, including in heavily pre-treated, TKI exposed patients.