FLT3-ITD Acute Myeloid Leukemia Patients Research Articles

Development of Epigenetic Modifiers with Therapeutic Potential in FMS-Related Tyrosine Kinase 3/Internal Tandem Duplication (FLT3/ITD) Acute Myeloid Leukemia and Other Blood Malignancies.

Blood cancers encompass a group of diseases affecting the blood, bone marrow, or lymphatic system, representing the fourth most commonly diagnosed cancer worldwide. Leukemias are characterized by the dysregulated proliferation of myeloid and lymphoid cells with different rates of progression (acute or chronic). Among the chronic forms, hairy cell leukemia (HCL) is a rare disease, and no drugs have been approved to date. However, acute myeloid leukemia (AML) is one of the most aggressive malignancies, with a low survival rate, especially in cases with FLT3-ITD mutations. Epigenetic modifications have emerged as promising strategies for the treatment of blood cancers. Epigenetic modulators, such as histone deacetylase (HDAC) inhibitors, are increasingly used for targeted cancer therapy. New hydroxamic acid derivatives, preferentially inhibiting HDAC6 (5a-q), were developed and their efficacy was investigated in different blood cancers, including multiple myeloma (MM), HCL, and AML, pointing out their pro-apoptotic effect as the mechanism of cell death. Among the inhibitors described, 5c, 5g, and 5h were able to rescue the hematopoietic phenotype in vivo using the FLT3-ITD zebrafish model of AML. 5c (leuxinostat) proved its efficacy in cells from FLT3-ITD AML patients, promoting marked acetylation of α-tubulin compared to histone H3, thereby confirming HDAC6 as a preferential target for this new class of hydroxamic acid derivatives at the tested doses.

FLT3-ITD Induces Immune Escape in AML Via up-Regulating CD47 Expression and Decreased Phagocytic Ability of Macrophages

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and confer a poor prognosis. FLT3 inhibitors such as Quizartinib could prolong survival. However, very few relapsed patients are cured. Little attention has been paid to the characteristics of the immune microenvironment in FLT3-ITD AML. Within the bone marrow, Flt3/ITD animals demonstrated significantly increased dendritic cells and T cells levels compared to Flt3/WT mice, which suggested that FLT3-ITD can modulate the immune microenvironment in AML. Here, we found that FLT3-ITD cell lines (MV-4-11 and MOLM-13) induced less LDH release compared to FLT3-WT cell lines (HEL and K562) ( Figure a), indicating less macrophage killing. CD47 gene is an integrin related protein, which acts as an “anti phagocytosis” signal, and it interacts with signal-regulatory protein alpha (SIRPalpha) on macrophages, so that tumor cells expressing CD47 can escape being destroyed by macrophages. Further Flow Cytometry results showed that the CD47 Mean fluorescent intensity (MFI) of the FLT3-ITD group was significantly higher than those of the control group ( Figure b). Therefore, we can ask a hypothesis that FLT3-ITD in tumor cells may leads to decreased phagocytic capacity of macrophages and immune tolerance. To test our hypothesis, the FLT3 wildtype (FLT3-OE, FLT3 over expression) or ITD mutant (FLT3-ITD) stable overexpression HEL/K562 cells were constructed by means of lentiviral transfections. In the FLT3-ITD group, the MFI levels of CD47 was higher than FLT3-OE group and FLT3 NC (FLT3 Normal control) group ( Figure c and d). Green-fluorescent (CFSE-stained) Hel and K562 were then added to cultures of macrophages induced by Peripheral Blood Mononuclear Cell stained with the red dye PKH26 (ratio tumor cell:macrophages, 5:1). Flow Cytometry results showed FLT3-ITD decreased the Phagocytic Capacity of Macrophages ( Figure e and f). The TRANSFAC database was used to predict the transcription factor binding sites of the CD47 promoter. We found that HOXB5 may regulate the expression of CD47 at the transcriptional level. Compared with the FLT3-NC and FLT3-OE group, the relative expression level of the CD47 gene in the FLT3-ITD group was significantly upregulated ( Figure g, p < 0.05). To ascertain the mechanism that HOXB5 regulated CD47, we analysed the location CD47 gene on the chromosome, as well as the sequence of promoter and first non-encoding exon of HOXB5 (−2000 ~ 100 bp) by Jaspar online tool (http://jaspar.genereg.net/). Mechanistically, Dual luciferase reporter gene assay and ChIP experiments suggest HOXB5 directly activate CD47( Figure h and i). Compared to the monotherapy group, Combination therapy with CD47 inhibitor and Quizartinib (AC220) significantly promoted the phagocytic ability of M1-like macrophages against MOLM13 cells ( Figure j and k). The mouse model of AML was constructed by injection of 5x106 FLT3-ITD cells via the tail vein. Mouse models further demonstrate that combination therapy with CD47 inhibitor and Quizartinib significantly reduces the tumor burden and improves survival rate. We first revealed that FLT3-ITD can up-regulates CD47 to promote immune escape of macrophages. These evidences indicates that combination therapy with CD47 inhibitor and FLT3-ITD inhibitor can be a promising therapeutic method for impressive treatment of FLT3-ITD AML patients.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.

Clinical study of induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation in the treatment of FLT3-ITD(+) acute myeloid leukemia with normal karyotype

Objective: To assess the efficacy of induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of FLT3-ITD(+) acute myeloid leukemia (AML) with normal karyotype. Methods: The clinical data of FLT3-ITD(+) AML patients with normal karyotype in the First Affiliated Hospital of Nanjing Medical University from Jan 2018 to March 2021 were retrospectively analyzed. Results: The study included 49 patients with FLT3-ITD(+)AML, 31 males, and 18 females, with a median age of 46 (16-59) years old. All patients received induction chemotherapy, and 24 patients received sequential allo-HSCT (transplantation group) . The median follow-up time was 465 days, the one-year overall survival (OS) from diagnosis was (70.0 ± 7.4) %, and one-year disease-free survival (DFS) was (70.3±7.4) %. The one-year OS was significantly different between the transplantation group and the non-transplantation group [ (85.2 ± 7.9) % vs (52.6 ± 12.3) %, P=0.049]. but one-year DFS [ (84.7 ± 8.1) % vs (55.2 ± 11.9) %, P=0.061] was not. No significance was found in one-year OS between patients with low-frequency and high-frequency FLT3-ITD(+) (P>0.05) . There were 12 patients with high-frequency FLT3-ITD(+) in the transplantation and the non-transplantation groups, respectively. The one-year OS [ (68.8 ± 15.7) % in the transplantation group vs (26.2 ± 15.3) % in the non-transplantation group, P=0.027] and one-year DFS [ (45.5 ± 21.3) % in the transplantation group vs (27.8±15.8) % in the non-transplantation group, P=0.032] were significantly different between the two groups. Conclusion: Induction chemotherapy followed by allo-HSCT can enhance the prognosis of FLT3-ITD(+) patients, particularly those with FLT3-ITD high-frequency mutation.

Real-World Efficacy Outcomes of FLT3-ITD Acute Myeloid Leukemia Patients Treated with Midostaurin in Combination with Intensive Induction

Acute myeloid leukemia (AML) patients harboring FLT3-ITD mutations are a high-risk patient subset owing to the increased risk of relapse and inferior overall survival (OS) associated with FLT3-ITD.1 While FLT3-ITDmut patients, following receipt of induction, attain comparable remission rates to patients without FLT3-ITD, these patients, especially those with higher allelic ratios are more likely to experience relapse, even after being referred to allogeneic transplantation.2,3 This has led the field to focus on the development of specific inhibitors to FLT3 with the anticipation that these agents would improve outcomes for FLT3-ITDmut patients.

Single-Cell Proteomic Interrogation of FLT3-ITD Leukemia Compartment Revealsdistinct Leukemia Cell Populations Resistant to Quizartinib, Decitabine and Venetoclax

Triplet therapy (TT) combining Quizartinib(Quiz), Decitabine(Dec) and Venetoclax(Ven) is highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates (Yilmaz M, ASCO 2022).Signaling mutations, particularly those involving RAS/MAPK pathway, have emerged as key contributors to primary and secondary resistance to TT. Lack or loss of response to TT is not limited to presence or emergence of clones with signaling mutations and disease relapse occurs in FLT3-ITD AML patients with a variety of other mutations. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by therapy pressure, and aid in identifying proteomic correlates of response and therapeutic vulnerabilities. To this end, we performed comprehensive single-cell proteomic analysis of serially collected samples (n:162) from patients treated with TT (n:14) and doublet (n:10, DEC+Quiz) therapy, using CyTOF and interrogated leukemia phenotypic profiles, apoptotic networks, signaling pathways, differentiation states and trajectories. First, we performed unsupervised analyses to identify leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner, interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features that may correlate with clinical response and assessed whether distinct mutation profiles are associated with specific proteomic profiles that may confer resistance to therapy. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators across leukemia landscapes and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC), generally situated in close proximity to leukemia cells in UMAPs, lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells (Figure 1). Due to their contrasting apoptosis regulatory networks, we observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Of note, leukemia cell compartments harbor distinct differentiation hierarchies, the frequencies of which varied across patients. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered and reshaped the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutation diversified the leukemia landscape into a diverse continuum of differentiation states. Remarkably, we observed that RAS/MAPK mutations in FLT3-ITD AML overcame differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells(Figure1). Importantly, we observed that transitional and CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs preferentially persisted at D28, had active signaling pathways and expressed CD36, which indicates distinct metabolic programs could ensure survival under therapy pressure and also had lower levels of BCL2.The presence of CD34+ leukemia cells at D28, adapted under therapy pressure with persistent signaling at D28, both in CD34+ leukemia or MCs, was indicative of poor clinical outcomes. Conclusion: Single-cell proteomic assessment of FLT3-ITD AML treated with Quiz/Dec/Ven (TT) identifies a unique mode of resistance and proteomic features of surviving cells at D28, and elucidates how RAS/MAPK mutations diversify the leukemia proteomic landscape and modulate the architectural organization which eventually gives rise to emerging clones resistant to therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

FLT3 Inhibitors Induce p53 Instability Due to Increased Interaction between p53 and MDM2 By Inhibiting STAT5 Phosphorylation in Acute Myeloid Leukemia

Frequently mutated in Acute myeloid leukemia (AML), FLT3 is considered as one of the favorable targets for treatment. The FLT3 internal tandem duplication (ITD) mutation enhances kinase activity and causes hyperactivation of downstream signal transduction. Several small molecule FLT3 inhibitors have developed, but their clinical efficacy is limited due to generation of drug resistance. In this study, we define a new mechanism of drug resistance toward tyrosine kinase inhibitors (TKIs). Initially, we found a rapid decrease in the protein level of tumor suppressor p53 in FLT3-ITD-positive MV4-11 and MOLM13 cells and peripheral blood mononuclear cells (PBMCs) from FLT3-ITD AML patients upon treatment with TKIs including sorafenib, sunitinib and quizartinib. The decrease is not caused by changes in mRNA expression as revealed by qPCR analyses but rather by accelerated protease degradation because the p53 protein was stabilized by proteasome inhibitor MG132. Furthermore, treatment of cells with RG7388, a potent disruptor of p53 and MDM2 interaction, prevented the TKI-induced p53 loss. Since MDM2 is the most important E3 ligase responsible for ubiquitination of p53, the data suggest that TKIs may lead to the degradation of p53 by promoting ubiquitination. Indeed, ubiquitination assays verified that TKIs promoted K48 poly-ubiquitination of p53. Previous studies have demonstrated that activations of FLT3 downstream signaling components such as ERKs and Akt reduce p53 protein stability through ubiquitination by activating MDM2. It is somewhat unexpected that inhibition of FLT3-ITD and its downstream signaling pathways also resulted in decreased p53 stability due to increased ubiquitination. We treated FLT3-ITD-containing cells with specific ERK, AKT and STAT5 inhibitors. Interestingly, while inhibition of ERKs and AKT had no significant effect on the stability of p53, STAT5 inhibition resulted in a reduced level of p53 accompanied by increased K48 poly-ubiquitination. We further analyzed the interaction of p53 with MDM2 in AML cells by using immunoprecipitation. The results showed that the p53-MDM2 interaction was significantly enhanced after treatment with TKIs and STAT5 inhibitors, which was diminished in the presence of RG7388. Subcellular fractionation revealed the presence of p53 and STAT5 in both nucleus and cytoplasm. Treatment of cells with TKIs resulted in a decreased level of p53 and STAT5 in the nucleus, and immunoprecipitation of nuclear proteins with a p53 antibody revealed a reduced association of p53 with STAT5. Taken together, the data suggest that FLT3 inhibitors inhibited nuclear translocation of STAT5 and reduced its interaction of p53 thereby facilitating p53/MDM2 interaction and subsequent ubiquitination and degradation of p53. This study reveals a novel mechanism by which drug resistance to TKIs may occur and further support the use of MDM2/p53 interaction inhibitors in combination with TKIs for treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Pim Kinase Inhibitor Enhances FLT3 Inhibitor Efficacy through GSK-3β Activation and GSK-3β-Mediated Proteasomal Degradation of c-Myc

BACKGROUNDInternal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc.METHODSBa/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry.RESULTSTreatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment.CONCLUSIONSConcurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

Midostaurin in combination with chemotherapy is most effective in patients with acute myeloid leukemia presenting with high FLT3‐ITD allelic ratio who proceed to allogeneic stem cell transplantation while in first complete remission

Midostaurin, a multikinase and FLT3 inhibitor, is the first non-chemotherapy agent approved and widely adopted for the treatment of FLT3-ITD acute myeloid leukemia (AML). Yet, its role in improving survival of patients referred to allogeneic stem cell transplantation (allo-SCT) in first complete remission (CR1) needs to be defined. This multicenter study retrospectively evaluated the outcome of 119 FLT3-ITD AML patients [59 (49.6%) males and 60 females] intensively treated between 2015 and 2019 at five Israeli centers. In our cohort, allo-SCT in CR1 was widely implemented (47%) and patient stratification was based on the current allelic ratio (AR) cutoff of 0.5. Ninety-eight patients (82.3%) achieved CR1/CR with incomplete count recovery (CRi). Death during induction was reported in 7 (5.9%) patients. In multivariate analysis, midostaurin use and allo-SCT in CR1 were the most significant factors affecting overall survival (OS). Midostaurin incorporation in chemotherapy regimens significantly improved CR+CRi rates (P=.002), reduced relapse rates (P=.02), and was remarkably advantageous for high-AR patients (2-year OS 82%). In low-AR patients, the midostaurin effect was much less prominent. Our results demonstrate benefits of midostaurin incorporation in intensive chemotherapy regimens, particularly for high-AR AML patients to whom it should be offered along with allo-SCT in CR1.

Real-World Outcomes of Patients with Refractory or Relapsed FLT3-ITD Acute Myeloid Leukemia: A Toulouse-Bordeaux DATAML Registry Study.

Two recent phase 3 trials showed that outcomes for relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) patients may be improved by a single-agent tyrosine kinase inhibitor (TKI) (i.e., quizartinib or gilteritinib). In the current study, we retrospectively investigated the characteristics and real-world outcomes of R/R FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) patients in the Toulouse-Bordeaux DATAML registry. In the study, we included 316 patients with FLT3-ITD AML that received intensive chemotherapy as a first-line treatment. The rate of complete remission (CR) or CR without hematological recovery (CRi) was 75.2%, and 160 patients were R/R after a first-line TKI-free treatment (n = 294). Within the subgroup of R/R patients that fulfilled the main criteria of the QUANTUM-R study, 48.9% received an intensive salvage regimen; none received hypomethylating agents or low-dose cytarabine. Among the R/R FLT3-ITD AML patients with CR1 durations < 6 months who received intensive TKI-free treatment, the rate of CR or CRi after salvage chemotherapy was 52.8%, and these results allowed a bridge to be transplanted in 39.6% of cases. Finally, in this QUANTUM-R standard arm-matched cohort, the median overall survival (OS) was 7.0 months and 1-, 3- and 5-year OS were 30.2%, 23.7% and 21.4%, respectively. To conclude, these real-world data show that the intensity of the second-line treatment likely affects response and transplantation rates. Furthermore, the results indicate that including patients with low-intensity regimens, such as low-dose cytarabine or hypomethylating agents, in the control arm of a phase 3 trial may be counterproductive and could compromise the results of the study.

Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736

Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.

A Phase II Single-Arm Open-Labeled Study Evaluating Combination of Quizartinib and Omacetaxine Mepesuccinate (QUIZOM) in Newly Diagnosed or Relapsed/Refractory AML Carrying FlT3-ITD

Objectives: Acute myeloid leukemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine Kinase 3 (FLT3) is associated with high relapse risk and adverse outcome. This single-arm, phase 2 study evaluated efficacy and safety of combination treatment with FLT3 inhibitor quizartinib and protein translation inhibitor omacetaxine mepesuccinate (OME), referred herein QUIZOM - in elderly patients with newly diagnosed FLT3-ITD AML or young patients with relapsed/refractory (R/R) diseases, whose outcome is hitherto dismal. Methods: R/R FLT3-ITD AML patients ≥ 18 years old or newly diagnosed patients ≥ 65 years old were recruited. Treatment comprised quizartinib 30 mg daily and OME [2 mg daily for 7 (first course) or 5 days (second course onwards) every 21-28 days] until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). Quizartinib was given as post HSCT maintenance after engraftment at doses ranging from 30 mg twice weekly to 30 mg daily, depending on blood counts. Primary endpoint was composite complete remission (CRc) defined by CR+CRi (CR with incomplete haematological recovery). Secondary endpoints were leukaemia-free (LFS) and overall survival (OS). Molecular responses were evaluated by FLT3-ITD based on PCR and NPM1 variant allele frequency (VAF) based on digital PCR (ddPCR). Mutation profiling was performed by MiSeq Next Generation Sequencing (NGS) based on IDT xGen Lockdown probes custom panel of 67 genes or TruSight myeloid panel of 54 genes. Results: Twenty-nine patients (R/R case=22; newly diagnosed case=7) were recruited between November 2017 and July 2019. Their clinicopathologic characteristics were shown in Table 1. Twenty-seven patients completed at least one course of QUIZOM (median= 2 courses, range: 1-20 courses) of whom 22 (R/R=19; newly diagnosed=3) achieved CR/CRi [CR=2 (7%); CRi=20 (74%), CRc=22 (81%)], 3 patients showed partial remission (PR) and 2 patients did not respond. All 6 patients who had prior FLT3 inhibitors (sorafenib or midostaurin) achieved CR/CRi. Median LFS and OS of CR/CRi group were 5.2 and 11.07 months (Figure 1). Seven patients received allogeneic HSCT of whom all have remained in remission post HSCT as of 31 July 2019 (OS 6.43 to 19.8 months) (Figure 1). Deep molecular responses (DMR) as defined by FLT3-ITD VAF ≤0.1% and NPM1 VAF ≤ 0.001% could be accomplished in 78% and 27% patients. Adverse effects associated with QUIZOM were primarily haematological and non-haematologic adverse effects were scarce. Two patients succumbed before commencement and on day 1 of QUIZOM due to pneumonia and intracranial haemorrhage. None of recurrently mutated genes was significantly associated with treatment responses in this cohort. Conclusion: QUIZOM is effective and safe for newly diagnosed and R/R FLT3-ITD AML. Acknowledgements: Li Shu Fan Medical Foundation, S.K. Yee Medical Foundation, Croucher Foundation, LKS Faculty of Medicine, University of Hong Kong, Daiichi Sankyo Inc. Disclosures No relevant conflicts of interest to declare.

Role of HDAC8 Upregulation in Resistance of FLT3-ITD AML Stem Cells to FLT3 Inhibitors

The FLT3-ITD mutation occurs in 25-30% of acute myeloid leukemia (AML) incidence, and is associated with higher relapse rate and inferior survival. FLT3 inhibitors have shown potent antileukemic effect, but the responses are not sustained in most FLT3-ITD AML patients. Histone deacetylases (HDACs) are crucial for cancer stem cell maintenance through epigenetically regulating gene expression, modulating the function of non-histone proteins via post-translational modification, and maintaining genome stability. Here we investigated the potential role of HDACs in FLT3 inhibitors resistance and FLT3-ITD AML stem cells maintenance. We firstly evaluated mRNA expression alterations of 11 HDACs in FLT3-ITD+ MV4-11 and MOLM-13 AML cells upon FLT3 inhibitor AC220 (Quizartinib) treatment, and found that HDAC8 was the most significantly upregulated gene. Inhibition of FLT3 signaling via siRNA-mediated knockdown of FLT3 also resulted in increased expression of HDAC8. Treatment of primary FLT3-ITD+ CD34+ AML cells with AC220 confirmed HDAC8 upregulation. Targeting HDAC8 using specific HDAC8 inhibitor 22d or knockdown of HDAC8 readily resulted in significant apoptosis of cell lines and primary AML samples, and inhibited colony growth and replating activity in primary FLT3-ITD+ CD34+ AML cells. Moreover, these antileukemic effects were even more pronounced upon 22d and AC220 combination. To explore the mechanism of HDAC8 mediated FLT3 inhibitor resistance, we investigated transcriptomic change of MV4-11 cells upon 22d treatment by RNA-seq. KEGG pathway analysis revealed that the p53 signaling pathway was significantly upregulated. Upstream regulator prediction by Ingenuity pathway analysis also showed p53 as the key activated upstream transcription factor. Western blot analysis on acetylated p53 (Lys382) demonstrated that targeting FLT3 signaling had little effect on the p53 acetylation, while suppression of HDAC8 markedly increased p53 acetylation, and this effect was even more significant when both FLT3 and HDAC8 were inhibited simultaneously. Knockdown of p53 could partially rescued FLT3-ITD AML cells from 22d and/or AC220 induced apoptosis, and offset the inhibitory effect on growth and colony formation. We next studied the mechanism of HDAC8 upregulation upon FLT3 signally inhibition. We found FOXO1 and FOXO3 (FOXOs) binding motifs in HDAC8 promoter. FLT3 signaling inhibition promoted the binding of FOXOs to HDAC8 promoter. Knockdown both FOXOs abrogated HDAC8 upregulation upon AC220 treatment, and caused growth inhibition and apoptosis of FLT3-ITD AML cells. Finally, to translate our experimental results into clinical usage, we tested the in vivo effect of 22d, AC220, and the combination of both on patient-derived xenograft (PDX) model engrafted with four primary FLT3-ITD AML samples. 22d or AC220 treatment reduced the percentage and total number of human CD45+ leukemic cells in murine bone marrow, and prolonged the survival of leukemic mice. The combination of 22d and AC220 significantly reduced AML cell engraftment compared with either 22d or AC220 alone, and further elongated the survival period. Human CD34+ cells in BM were markedly reduced in mice treated with 22d or AC220 alone, with further reduction seen in combination. Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment compared with AC220 treatment alone, indicating reduced LSC capacity of residual cells. These results show that combination of 22d and AC220 enhances targeting of primitive AML stem cells in vivo. In conclusion, upon FLT3 inhibitor treatment, FLT3-ITD AML cells may upregulate the expression of HDAC8 through FOXOs activation, which in turn inhibit the function of tumor suppressor gene p53, and promote the maintenance of FLT3-ITD AML stem cells and drug resistance. Targeting FLT3 and HDAC8 simultaneously may be promising approach to eliminating FLT3-ITD AML stem cells, and to cure this subset of leukemia. Disclosures No relevant conflicts of interest to declare.

Single Cell DNA Sequencing Identifies Combinatorial Mutation Patterns and Clonal Architecture in Myeloid Malignancies

Genomic studies of myeloid malignancies (MM), including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN) and myelodysplasia (MDS), identified mutations with different allele frequencies. Recent studies of clonal hematopoiesis (CH) discovered a subset of MM disease alleles, while other alleles are only observed in overt MM. These observations suggest an important pathogenetic role for the chronology of mutational acquisition. Although bulk sequencing informs prognostication, it cannot distinguish which mutations occur in the same clone and cannot offer definitive evidence of mutational order. Delineation of clonal architecture at the single cell level is key to understanding how the sequential/parallel acquisition of somatic mutations contributes to myeloid transformation. In order to elucidate the clonal structure of MM, we designed a custom single cell 109 amplicon panel of the most frequently mutated amplicons in 50 MM genes using the Mission Bio Tapestri v2 platform. Viable cells were sorted from 90 samples from 78 patients with CH, AML, and MPN/post-MPN AML followed by single cell amplification/sequencing. Mutation calls were filtered based on read depth, quality, and alleles genotyped per cell. We reconstructed a random distribution of clones by permuting genotype calls across cells and generated empirical p values for each clone. To identify dominant clones, we used a Poisson test to determine clones were significantly enriched compared to the mean clone size. Clones with significant p-values (p &amp;lt;0.05) were used to generate plots of clonal architecture of each sample (Figure 1A). Despite significant clonal complexity, the majority of MM patients (80%;72/90) present with one (51/90; 56.7%) or two (21/90; 23.3%) dominant clones. These data show there are specific genotypic combinations which lead to clonal dominance with increased fitness relative to other clones and/or suppression of minor clones by dominant clone(s). We next investigated whether specific molecularly defined AML subtypes had increased clonal complexity. FLT3-ITD mutant AML samples had a significantly greater number of clones (p &amp;lt; 0.002) compared to AML samples with multiple epigenetic modifier mutations. Similar findings were not observed when comparing AML samples with epigenetic mutations to RAS pathway mutant samples. We next investigated whether specific mutations were likely to co-occur/be mutually exclusive at a single cell level. We observed evidence of oligoclonality in CH, including parallel acquisition of DNMT3A mutations and clones with multiple mutations in the absence of progression to MM. By contrast, in MM the dominant clone(s) almost always harbored multiple epigenetic modifier mutations, suggesting cooperative epigenetic remodeling in myeloid transformation. Mutations in signaling effectors (FLT3-ITD/TKD; RAS/RAS) were mutually exclusive. We observed distinct FLT3-mutant clones in FLT3-mutant AML patients and parallel acquisition of different RAS pathway mutations. We used this data to develop clonal architecture trees in each patient, giving us a definitive picture of mutational acquisition and transformation at a single cell level. We calculated a Shannon diversity score and observed an increase in clonal complexity with disease evolution; CH samples had the lowest clonal diversity and FLT3-ITD AML patients the highest clonal diversity (Figure 1B). We extended our findings by combining cell surface marker assessment and single cell mutational analysis. Patient samples were stained with an antibody cocktail of 6 oligo-conjugated antibodies with barcode tags prior to single cell sequencing, which allowed simultaneous acquisition of single cell immunophenotypic and genotypic data. This allows us to identify distinct populations of stem/progenitor cells with distinct clonal/mutational repertoires (Figure 1C). Additional data will be presented with this novel approach, which allows us to combine an assessment of stem/progenitor cell frequency with genetic data. This includes studies of CD34+ and CD34- AML, which show striking differences in mutational representation in different stem/progenitor compartments. In summary, our studies of clonal architecture at a single cell level provide us novel insights into the pathogenesis of myeloid transformation and give us new insights into how clonal complexity contributes to disease progression. Disclosures Ooi: Mission Bio: Employment, Equity Ownership. Mendez:Mission Bio: Employment, Equity Ownership. Carroll:Janssen Pharmaceuticals: Consultancy; Incyte: Research Funding; Astellas Pharmaceuticals: Research Funding. Papaemmanuil:Celgene: Research Funding. Viny:Mission Bio: Other: Sponsored travel; Hematology News: Membership on an entity's Board of Directors or advisory committees. Levine:Roche: Consultancy, Research Funding; Amgen: Honoraria; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Gilead: Consultancy; Celgene: Consultancy, Research Funding.

Abstract 2475: FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia

Abstract Introduction. Acute Myeloid Leukemia (AML) is the most common acute Leukemia among adults with a poor overall prognosis. With over 20,000 cases in the US alone, about a third of AML patients pertain to an important oncogenic driver composed of internal tandem duplication (ITD) somatic mutations in exons 14 and 15 of the FMS-like tyrosine kinase 3 (FLT3) gene. Detecting the presence of measurable residual disease in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. Yet, despite its importance, the ability to detect FLT3-ITD mutations is hampered by the limited sensitivity of conventional PCR-based assays in general and in Next Generation Sequencing-based assays, in part due to the difficulty to align ITD-containing reads to the reference genome. Methods. In order to facilitate the analysis of AML samples, we developed FLT3-Explorer, a comprehensive bioinformatics pipeline composed of open source software, combined with in-house algorithms to accurately detect low levels of FLT3-ITD mutations. The input of the pipeline is unmapped reads in fastq format. The pipeline then searches for either ITD sequences previously observed in our extensive database of historical data, or a de-novo detection procedure is used to determine mutation sequences of variable lengths within the FLT3 gene. To assess the performance of FLT3-Explorer, we considered a cohort of clinical samples from bone marrow and peripheral blood, as well as cell lines. For sample collection, genomic DNA was isolated, and polymerase chain reaction was performed using primers flanking exons 13 and 15 to amplify the FLT3 gene. DNA was then sequenced using NGS Illumina sequencing. Results were validated using fragment size analysis by Capillary Electrophoresis assays. Also, to determine the limits of detection, various dilutions were performed on cell lines. Furthermore, to assess the performance of our pipeline, we performed a comparison with other FLT3-ITD programs publicly available. Summary. Results from our analysis showed that our method identifies FLT3-ITD mutations of variant allele fraction as low as 0.003%. Also, we found that our pipeline can detect as many FLT3-ITD mutants as other methods, or more, when the mutant signal is considerably low. Conclusions. FLT3-Explorer constitutes a powerful solution for low FLT3-ITD detection signal of mutant sequences, of diverse lengths, that can be used on FLT3-ITD AML patients to track disease progression and relapse. Citation Format: Christian Laing, Wenge Shi, Reinhold Pollner. FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2475.

Analysis of induction efficacy and prognostic factors in FLT3-ITD positive acute myeloid leukemia in the real world

目的探讨真实世界FLT3-ITD突变阳性急性髓系白血病（FLT3-ITD+AML）诱导治疗的疗效及预后影响因素。方法收集2013年1月至2016年12月收治的初发FLT3-ITD+AML患者114例，了解患者初诊时生物学特征，采用化疗方案进行诱导治疗，随访病例，并对其缓解率、预后等进行分析。结果114例FLT3-ITD+AML患者初诊时分子学检测发现伴随预后良好基因突变52例（NPM1突变46例，RUNX1-RUNX1T1 5例，CEBPA双突变1例）；其他类型FLT3-ITD+AML 62例。所有患者均至少完成1个疗程的诱导治疗并进行了疗效评估，1个疗程完全缓解（CR）率为50.0%（57/114），2个疗程CR率为72.5%（74/104）。52例伴随预后良好基因突变的FLT3-ITD+AML患者1个疗程CR率为67.3%（35/52），2个疗程CR率为83.3%（40/48）；62例其他类型FLT3-ITD+AML患者1个疗程CR率为35.5%（22/62），2个疗程CR率为64.8%（35/54）；两组CR率差异有统计学意义（P<0.05），显示FLT3-ITD伴随预后良好基因突变的AML患者治疗效果相对较好。对于其他类型FLT3-ITD+AML 1个疗程未缓解的患者，随后采用索拉非尼联合化疗的9例患者中6例达到CR，缓解率为66.7%；采用常规化疗方案的23例患者中7例达到CR，缓解率为30.4%，两者之间差异有统计学意义（χ2=4.47，P<0.05），显示索拉非尼联合化疗方案可以显著提高FLT3-ITD+AML不缓解患者的CR率。索拉非尼联合化疗与常规方案化疗患者总生存和无病生存比较，差异均无统计学意义（P值分别为0.641和0.517）。结论FLT3-ITD+AML患者整体预后不良，通过索拉非尼联合化疗的方案对不伴随预后良好基因突变的FLT3-ITD+AML进行分层治疗可以提高疗效。

8-chloro-adenosine activity in FLT3-ITD acute myeloid leukemia.

Nucleoside analogs represent the backbone of several distinct chemotherapy regimens for acute myeloid leukemia (AML) and combination with tyrosine kinase inhibitors has improved survival of AML patients, including those harboring the poor-risk FLT3-ITD mutation. Although these compounds are effective in killing proliferating blasts, they lack activity against quiescent leukemia stem cells (LSCs), which contributes to initial treatment refractoriness or subsequent disease relapse. The reagent 8-chloro-adenosine (8-Cl-Ado) is a ribose-containing, RNA-directed nucleoside analog that is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. In this report, we demonstrate antileukemic activities of 8-Cl-Ado in vitro and in vivo and provide mechanistic insight into the mode of action of 8-Cl-Ado in AML. 8-Cl-Ado markedly induced apoptosis in LSC, with negligible effects on normal stem cells. 8-Cl-Ado was particularly effective against AML cell lines and primary AML blast cells harboring the FLT3-ITD mutation. FLT3-ITD is associated with high expression of miR-155. Furthermore, we demonstrate that 8-Cl-Ado inhibits miR-155 expression levels accompanied by induction of DNA-damage and suppression of cell proliferation, through regulation of miR-155/ErbB3 binding protein 1(Ebp1)/p53/PCNA signaling. Finally, we determined that combined treatment of NSG mice engrafted with FLT3-ITD + MV4-11 AML cells with 8-Cl-Ado and the FLT3 inhibitor AC220 (quizartinib) synergistically enhanced survival, compared with that of mice treated with the individual drugs, suggesting a potentially effective approach for FLT3-ITD AML patients.

Monitoring of FLT3 Phosphorylation and FLT3 Ligand Levels in Patients with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) Treated with Midostaurin within the AMLSG 16-10 Trial of the German-Austrian Study Group

Monitoring of FLT3 Phosphorylation and FLT3 Ligand Levels in Patients with FLT3-ITD Mutated Acute Myeloid Leukemia (AML) Treated with Midostaurin within the AMLSG 16-10 Trial of the German-Austrian Study Group

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition

AbstractFLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation–driven leukemias.