FMS-like Tyrosine Kinase 3 Expression Research Articles

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

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

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

Proteolysis targeting chimeras (PROTACs) induce the ubiquitination and subsequent proteasomal degradation of targeted proteins. Numerous PROTACs have emerged as promising drug candidates for various disease-related proteins. This study investigates PROTACs targeted to degrade anaplastic lymphoma kinase (ALK) fusion proteins, which are implicated in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. We recently reported the development of a gilteritinib-warheaded PROTAC to target and degrade the Fms-like tyrosine kinase 3 (FLT3) protein. Gilteritinib is a tyrosine kinase inhibitor that targets FLT3, and recent studies have revealed that it also functions as an ALK inhibitor. We conducted a structure–activity relationship (SAR) study and expanded the range of target proteins for gilteritinib-warheaded PROTACs to include echinoderm microtubule-associated protein-like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK, in addition to FLT3. Our SAR study utilized three types of ligands for E3 ligase— inhibitor of apoptosis protein (IAP), cereblon (CRBN), and von Hippel-Lindau (VHL)— in the PROTAC designs and we observed varied efficacy in the degradation of target proteins. The CRBN-based PROTAC effectively reduced the protein expression of FLT3, EML4-ALK, and NPM-ALK. The IAP-based PROTAC reduced expression of both FLT3 and EML4-ALK proteins but not that of NPM-ALK, while the VHL-based PROTAC was ineffective against all target proteins. Several ALK-targeted PROTACs have already been developed using CRBN or VHL as E3 ligase, but this is the first report of an IAP-based ALK degrader. The length of the linker structure utilized in PROTAC also had a significant effect on their efficacy and activity. PROTACs formed with shorter linkers demonstrated an enhanced degradation activity to target proteins compared with those formed with longer linkers. These findings provide valuable insight for the development of effective PROTACs to target and degrade ALK fusion proteins.

High FLT3 expression indicates favorable prognosis and correlates with clinicopathological parameters and immune infiltration in breast cancer.

Purpose: Breast cancer is a highly heterogeneous malignancy, seriously threatening female health worldwide and inducing higher mortalities. Few have the studies evaluated Fms-like TyrosineKinase-3 (FLT3) in prognostic risk, immunotherapy or any other treatment of breast cancer. Our study focused on investigating the function of FLT3 in breast cancer. Patients and methods: Based on transcriptome and methylation data mined from The Cancer Gene Atlas (TCGA), we explored the clinical features of FLT3 expression in 1079 breast cancer samples. RT-qPCR in cell lines and tissue samples was used to verify the expression difference of FLT3. Kaplan–Meier survival analysis and cox regression models were employed for screening of FLT3 with potential prognostic capacity. Subsequently, functional analysis of the co-expressed genes was conducted using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene-set enrichment analysis (GSEA). The correlation between FLT3 expression and tumor immune infiltration was jointly analyzed with estimate, ssGSEA, TIMER, and TISIDB. Then we employed checkpoint-related molecules, immunophenoscore (IPS), and tumor mutation burden (TMB) to assess the efficacy of immuno-checkpoint inhibitors (ICIs). Pearson correlation coefficient was employed to exam the association between DNA methylation and FLT3 expression. Results: FLT3 displays an elevated expression in breast cancer than normal pairs and is significantly associated with multiple clinical characteristics like age, menopause status, histological type, pathological stage, and molecular subtype as well as increased overall survival (OS). Additionally, FLT3 is a favorable independent prognostic factor. GO, KEGG, and GSEA suggested that FLT3 was associated with diversified immune-related features. FLT3 expression is correlated with the abundance of various immune cells namely CD4+T cell, CD8+ T cell, myeloid dendritic cell, and neutrophil as well as immune inhibitors especially CTLA4, which is positively correlated with FLT3 expression. Moreover, TMB displayed a negative correlation with FLT3 expression while IPS showed adverse tendency. Ultimately, the methylation of FLT3 downregulates the gene expression and closely binds to a few clinical parameters. Conclusion: FLT3 can be used for prognostic prediction and is relevant to immune infiltration in breast cancer. FLT3 may pave the way for future novel immunotherapies.

Detection of TET2 Mutation in Patients with De Novo Acute Myeloid Leukemia: A Mutation Analysis of 51 Iranian Patients.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease that is considered to originate from hematopoietic stem cells, which are characterized by impaired myelopoiesis and blast proliferation. TET oncogene family member 2 (TET2) mutations are frequent in myeloid malignancies and several studies have assessed the clinical importance of TET2 mutations. However, its frequency ratio has not yet been fully clarified. Method:Hence, our study was aimed to analyze TET2mut in patients with de-novo AML and their association with clinical, molecular characteristics and Nucleophosmin 1 (NPM1), Fms-like tyrosine kinase 3 (FLT3), CCAAT Enhancer Binding Protein Alpha (CEBPA) and Wilms’ tumor protein (WT1) gene expression. Fifty-one Iranian patients were screened by polymerase chain reaction (PCR) and direct sequencing to evaluate TET2 mutations frequency. Results:Out of all patients, 10 mutations in 8 patients (15.6%) were detected and closely associated with higher age and higher hemoglobin levels (p-value <0.05). Although FLT3, NPM1 and CEBPA gene expression did not show any significant correlation with TET2mut, cytogenetically normal acute myeloid leukemia (CN-AML) patients appear to bear TET2mut more frequently with lower platelet counts. Monocyte-lineages leukemia has seemed to be more linked with TET2mut in these patients. Conclusion:Our study suggests the frequency of TET2mut in our study (15.6%) is in line with previous studies and reveals the critical role of TET2 in myeloid transformation, especially in leukemia with monocytic subtypes.

A clinicopathological experience in acute myeloid leukemia: Effects of clinical data and status of FLT3, CEBPA and NPM1 on prognosis.

The purpose of the study was to analyze the expression of nucleophosmin (NPM1), CCAT/enhancer-binding protein alpha (CEBPA), and FMS-like tyrosine kinase 3 (FLT3) with immunohistochemistry and evaluate the relationship with clinicopathologic data with special emphasis on prognosis in bone marrow biopsy specimens diagnosed with acute myeloid leukemia (AML). Bone marrow biopsies of 104 patients who were diagnosed with AML were re-evaluated for diagnosis and subclassification. Immunohistochemically, anti-NPM1, anti-CEBPA, and anti-FLT3 antibodies were applied to slides prepared from formalin-fixed paraffin-embedded tissues. Sixty-three of these patients had their follow-up in our institutional hematology clinic and these patients' clinical, biochemical, and radiological data were obtained and analyzed from patient files. These data were analyzed with survival times statistically. Except for age, no significant effect of clinical data on prognosis was detected. Immunohistochemical results were also statistically compared with clinical data. No correlation was found between overall survival and disease-free survival with the expression of anti-CEBPA or anti-NPM1 antibodies. However, immunohistochemical reactivity for anti-FLT3 antibody was found to be a poor prognostic factor and statistically significant. Also, when the expression of FLT3 was analyzed with that of NPM1 or CEBPA, a correlation (dependent on the expression of FLT3) was found with disease-free survival. FLT3 is an independent prognostic factor for AML. CEBPA and NPM1 should be considered as good prognostic factors only in the absence of FLT3 abnormalities.

Relation between FMS-like tyrosine kinase 3 factor and hematological parameter in acute lymphoblastic leukemia patients by flow cytometry

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder that is caused by the clonal expansion of immature lymphoid cells with a high rate among children more than adults. FMS-like tyrosine kinase 3 (FLT3) is a cellular receptor belongs to the Class III receptor tyrosine kinase family. The main expression of FLT3 on bone marrow (BM) cells especially CD34+ hematopoietic stem cells, early progenitor cells, dendritic progenitor cells, and other cells of organs (brain, placenta, and testis). Activation of FLT3 results in increased cell proliferation, decreased cell apoptosis, and inhibition of differentiation of cells. This study aims to detect the expression of the FLT3 cluster of differentiation antigen 135 (CD135) in childhood B-ALL patients. Moreover, to correlate this expression with hematological parameters include a complete blood count and BM examination findings and clinical parameters. PATIENTS, MATERIALS AND METHODS: This study was conducted on 30 newly diagnosed pediatric ALL patients. Diagnosis of the disease was based on the blood film, BM examination findings, cytochemistry, and flowcytometry of peripheral blood (PB) and/or BM sample, 1 ml of PB and/or BM sample was collected in EDTA tubes for flowcytometry for detection of CD135. RESULTS: This study found that male patients were more than females with a male-to-female ratio (1.14:1) and a median age of 5 years. Most of the patients had a positive expression of the FLT3 receptor and according to NCI risk groups, 60% of patients fall in the standard risk and 40% in the high-risk group. There was a significant correlation between the level of FLT3 (CD135) and age but no significant correlation with hemoglobin, white blood count, platelets, and peripheral or BM blast percentage. CONCLUSION: In this study, the patients with positive FLT3 blast cells (which is a bad prognostic factor) were associated with good prognostic factors. This proves that FLT3 is an independent prognostic factor.

Homoharringtonine Synergized with Gilteritinib Results in the Downregulation of Myeloid Cell Leukemia-1 by Upregulating UBE2L6 in FLT3-ITD-Mutant Acute Myeloid (Leukemia) Cell Lines.

FMS-like tyrosine kinase 3 (FLT3) mutant acute myeloid leukemia (AML) occurs in approximately 30% of all AML patients and still has a poor prognosis. This study is directed to investigate gilteritinib in combination with homoharringtonine (HHT) on FLT3-ITD-mutant AML cell lines. In our study, we found that cell proliferation was dramatically suppressed by the combination of gilteritinib and HHT. This combination therapy decreased the mitochondrial membrane potential, finally inducing apoptosis. We demonstrated that gilteritinib downregulated the expression of FLT3 and downstream signaling, further decreased the mRNA level of myeloid cell leukemia-1 (Mcl-1). HHT and combination therapy could upregulate UBE2L6, which induced the degradation of Mcl-1 via ubiquitin-proteasome system. Knockdown of UBE2L6 could protect Mcl-1 from deprivation through the ubiquitin-proteasome system. These findings may provide a novel theoretical basis for the treatment of AML patients with FLT3-ITD mutations.

DOCK2 Knockdown Mitigates Disease Progression in Murine Models Harboring a FLT3/ITD Mutation

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts in the bone marrow and other tissues. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis.Previous work in the laboratory has demonstrated that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is also expressed in the leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity, and leads to decreased survival of leukemic cells that carry a FLT3/ITD mutation. This effect is seen in both in vitro studies and a mouse xenograft model, and is enhanced by treatment with cytarabine. Initial studies have shown that the anti-leukemic effect of DOCK2 KD may, in part, be due to reduced expression of both FLT3 and key DNA Damage Response (DDR) factors. In order to further investigate the relationship between DOCK2 and FLT3/ITD in hematopoietic neoplasms, we utilized mice that harbor a spontaneous Dock2 mutation (Dock2/Hsd). This mutation results in a substantial reduction in Dock2 mRNA and protein expression in hematolymphoid cells.Flt3/ITD mice develop a myeloproliferative neoplasm (MPN) that includes greatly increased spleen size. In contrast, the spleens of progeny of Flt3/ITD mice bred with Dock2/Hsd mice were greatly reduced in size. Flow cytometric analysis of the bone marrow also demonstrated that DOCK2 KD results in at least a partial reversal of the Flt3/ITD phenotype. Flt3/ITD mice characteristically demonstrate an increased fraction of immature hematopoietic stem/progenitor cells (Lin-Sca-1+c-KIT+ cells or LSKs); however, the fraction of LSKs in Flt3/ITD-Dock2/Hsd mice is similar to that seen in the marrow of WT mice. While the Flt3/ITD-Dock2/Hsd mice continued to show a disruption in the early to late Pro-B transition similar to that described in Flt3/ITD mice, the Flt3/ITD-Dock2/Hsd mice had a small but significant increase in the late Pre-B cell population, suggesting a less stringent block in B-cell development. Additionally, as compared with Flt3/ITD mice, the bone marrow of Flt3/ITD-Dock2/Hsd mice exhibited a decreased fraction of Mac1+Gr-1+ myeloid cells with a concomitant increase in Ter119+CD41- erythroid precursors, to levels that are indistinguishable from WT mice. These findings indicate that DOCK2 KD mitigates the myeloproliferative phenotype of Flt3/ITD mice.Since experiments using FLT3/ITD cell lines have suggested that DOCK2 KD may act by reducing expression of the FLT3 protein itself and also by inhibiting the DNA Damage Response, we evaluated these factors in the Flt3/ITD-Dock2/Hsd mice. Quantitative PCR of mouse whole bone marrow revealed that the mRNA levels of the Flt3 transcription regulators Meis1 and c-Myb were decreased in the Flt3/ITD-Dock2/Hsd mice, compared with Flt3/ITD mice, and the expression of Flt3 itself was markedly decreased as well. Moreover, reduced expression of key DDR factors including Chk1, Wee1, Rad51, Pim1, MSH2, MSH6 and MLH1 was also observed in Flt3/ITD-Dock2/Hsd mice.Finally, to assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine AML model that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. The Flt3/ITD-NHD13 mice develop AML with 100% penetrance and short latency; however, the expression of Dock2/Hsd in Flt3/ITD-NHD13 mice extended the median survival of these mice from approximately 110 days to over 155 days (and counting).These findings suggest that DOCK2 may cooperate with FLT3/ITD and play an important role in disease progression of FLT3/ITD leukemia. Thus, pharmacologic inhibition of DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Additionally, since DOCK2 KD results in decreased expression of DDR factors, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML. DisclosuresNo relevant conflicts of interest to declare.

CAR T Cells Targeting FLT3 on AML Confer Potent Anti-Leukemic Activity and Act Synergistically with the FLT3 Inhibitor Midostaurin

Background: FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein that is expressed on normal hematopoietic stem (HSC) and progenitor cells. FLT3 is also uniformly present on malignant blasts in acute myeloid leukemia (AML) providing a target for immunotherapy with chimeric antigen receptor (CAR)-modified T cells. FLT3 is a driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3-ITD) and other gain-of-function mutations in the intracellular tyrosine kinase domain. Accordingly, the FLT3 inhibitor midostaurin has recently been approved in combination with chemotherapy to treat FLT3-mutated AML. Here, we explored the antileukemia efficacy of FLT3 CAR-T cells against FLT3 wild-type (wt) and FLT3-ITD+ AML, alone and in combination with midostaurin to determine whether the two therapeutic modalities could be used synergistically, particularly in high-risk FLT3-ITD+ AML.Methods: A FLT3-CAR (BV10 scFv_IgG4 hinge_CD28_CD3ζ_EGFRt) was encoded in the lentiviral vector epHIV7 for gene-transfer into T cells of healthy donors and AML patients (n>6). CAR-T cell cytolytic activity was evaluated in FACS-/luminescence-based assays, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. Immunodeficient NSG mice were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR-modified or control T cells (CD4:CD8 ratio = 1:1). MOLM-13 cells were cultured in the presence of 10nM midostaurin and doses titrated up to 50nM to induce resistance (MOLM-13R).Results: We confirmed specific recognition and high-level cytolytic activity of CD8+ FLT3 CAR-T cells against a panel of AML cell lines that included THP-1 (FLT3 wt), MOLM-13 (FLT3-ITD+/-) and MV4;11 (FLT3-ITD+/+), as well as FLT3-ITD+ primary AML blasts obtained from multiple patients. Both CD8+ and CD4+ FLT3 CAR-T cells produced IFN-γ and IL-2, and underwent proliferation after stimulation with native AML cell lines and primary AML blasts.We then treated AML cell lines with midostaurin and detected a significant increase in FLT3 expression in MOLM-13 and MV4;11 by flow cytometry (delta MFI = 8986 and 1442, respectively, p<0.05), but not THP-1 cells, indicating that upregulation specifically occurred in FLT3-ITD+ AML. We sought to determine whether higher FLT3 expression on midostaurin-resistant AML target cells translated into superior antileukemia efficacy of FLT3 CAR-T cells. Indeed, we observed superior cytolytic activity, cytokine production and proliferation of FLT3 CAR-T cells after stimulation with MOLM-13R compared to native MOLM-13 cells (n=3 experiments; p<0.05). In contrast to FLT3, expression of CD33 and CD123 as alternative CAR target antigens was decreased on MOLM-13R.We confirmed upregulation of FLT3 occurred on native MOLM-13 AML cells in vivo during midostaurin treatment in NSG mice, and observed synergistic antileukemia efficacy of FLT3 CAR-T cells and midostaurin in combination therapy. Intriguingly, we detected higher frequencies of CAR-T cells in all mice that received the combination treatment, compared to mice that received FLT3 CAR-T cells alone. At the end of the observation period, all mice in the combination treatment group had achieved complete remission of AML from peripheral blood, bone marrow and spleen.Importantly, we also show that FLT3 CAR-T cells recognize normal HSCs and interfere with hematopoiesis in colony formation assays in vitro . However, midostaurin treatment did not lead to an increase of FLT3 on normal HSC consistent with their expression of wt FLT3.Summary: Collectively, our data demonstrate that FLT3 CAR-T cells mediate potent reactivity against FLT3 wt and FLT3-ITD+ AML cells in vitro and in vivo . We further demonstrate that FLT3 CAR-T cells and midostaurin act synergistically, i.e. midostaurin-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSC, the clinical use of FLT3 CAR-T cells is likely restricted to a defined therapeutic window prior to allogeneic HSC transplantation, followed by CAR-T cell depletion and hematopoietic reconstitution. Our data provide an operational model to augment the antileukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk for relapse with FLT3-negative AML variants that may otherwise develop under therapeutic pressure. DisclosuresNo relevant conflicts of interest to declare.

Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression

BackgroundMost patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30–40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells.MethodsRecombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD).ResultsSoluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo.ConclusionsOur data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

Studies on the anti-psoriasis effects and its mechanism of a dual JAK2/FLT3 inhibitor flonoltinib maleate

Psoriasis is a chronic, inflammatory autoimmune disease mediated by T cells, and characterized with abnormal proliferation and differentiation of keratinocytes, and inflammatory infiltration. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway has been identified to play essential roles in mediating various of biological processes, and is closely related to autoimmune diseases. Dendritic cells (DCs) are important antigen presenting cells and play an important regulatory role in T cells. The proliferation, differentiation and function of DCs are regulated by JAK and FMS-like tyrosine kinase 3 (FLT3) signal pathways. Flonoltinib maleate (FM), a high selectivity dual JAK2/FLT3 inhibitor with IC50 values of 0.8 nM and 15 nM for JAK2 and FLT3, respectively, was developed by our laboratory. Moreover, FM was a potent JAK2 inhibitor with 863-fold and 696-fold selectivity over JAK1 and JAK3, respectively. In this study, the anti-psoriasis activity of FM was evaluated both in vitro and in vivo. FM effectively inhibited the proliferation of HaCaT, the inflammatory keratinocyte induced by M5 and markedly suppressed the generation and differentiation of DCs from bone marrow (BM), and inhibited the expression of FLT3 in DCs in vitro. FM effectively inhibited the ear thickening and improved the pathological changes of the ear in interleukin (IL)-23-induced psoriasis-like acanthosis mouse model. Further in keratin 14-vascular endothelial growth factor (K14-VEGF) transgenic homozygous mice model, FM could obviously improve the psoriatic symptom and pathological changes, significantly inhibit the generations of Th1 and Th17 cells in the spleen, and the accumulations of DCs in the ears. FM could also significantly reduce the expression of various inflammatory factors both in C57BL/6 and K14-VEGF mice ears, and the serum of K14-VEGF mice. Mechanism revealed that FM effectively suppressed the phosphorylation of JAK2, STAT3 and STAT5 in inflammatory keratinocytes and the mice ears of C57BL/6 and K14-VEGF, as well as the phosphorylation of FLT3 in K14-VEGF mice ears. In conclusion, FM plays an excellent anti-psoriasis activity, including inhibiting keratinocyte proliferation and regulating inflammatory response through inhibiting JAK2 and FLT3 signaling pathway.

FLT3 Ligand-DM1 Conjugate Selectively Targets Acute Myeloid Leukemia Cells with FLT3 Expression

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm featured by impaired differentiation and uncontrolled proliferation of myeloid progenitors. Gain-of-function mutations of FMS-like tyrosine kinase 3 (FLT3) present in 30-40% of patients with AML. In addition, more than 90% of AML blasts aberrantly express FLT3, making FLT3 an attractive therapeutic target for AML. Currently, several small molecule tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved in the treatment of AML, but they need to be used in combination with chemotherapy because of their limited potency to eliminate leukemic cells as single agents, largely due to the development of secondary inhibitor-resistant FLT3 mutations. Therefore, novel therapeutic strategies targeting FLT3 are needed. In the present study, we developed a FLT3 ligand-emtansine drug conjugate (FL-DM1) that targeted FLT3-positive AML cells with high potency and selectivity. We expressed recombinant human FLT3 ligand (rhFL) in the periplasm of recombinant E. Coli. The protein was purified by a two-step purification system containing Ni-NTA and Phenyl Sepharose. Our purified rhFL was bioactive to stimulate phosphorylation of FLT3 and proliferation of THP-1 cells. Next, we conjugated purified rhFL and emtansine (DM1) with SPDP linker. Reducing and non-reducing SDS-PAGE revealed that rhFL and DM1 were successfully conjugated, evidenced by a band with higher molecular weight of the conjugation product. Previous studies show that DM1 is a drug preferentially targeting proliferating cells by depolymerizing microtubules through binding at the vinca binding site of tubulin. We found that FL-DM1 reserved the physiological function of FLT3 ligand to stimulate proliferation of AML cells by inducing phosphorylation of FLT3 and the downstream signaling protein AKT in immunoblot, potentially enhancing the potency of FL-DM1 to inhibit FLT3-positive AML cells. Furthermore, flow cytometry showed that the surface expression of FLT3 significantly decreased on cells treated by FL-DM1 within two hours, which indicated the internalization of FL-DM1/FLT3 complex on these FLT3-positive AML cells, providing a mechanism of FL-DM1 entering target cells. In cell viability assay, we found that FL-DM1 effectively inhibited FLT3-positive AML cells THP-1 and MV-4-11 with IC50 around 10-30 nM. Also, FL-DM1 induced apoptosis and cell cycle arrest at G2/M phase in these cells detected by flow cytometry. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are FLT3-negative erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. We found that FL-DM1 inhibited HCD-57 cells transformed by FLT3-ITD, but not parental HCD-57 cells without FLT3 expression, indicating the selectivity of FL-DM1 to target FLT3-positive AML cells. In conclusion, our data demonstrated that FL-based drug conjugate can serve as an effective drug to target FLT3-expressing AML cells. Further studies will focus on in-vivo evaluation of FL-DM1 in animal models, the production of uncleavable SMCC linked FL-DM1 with improved in-vivo pharmacokinetic properties, and screening of FL muteins-DM1 conjugates with desired pharmacological properties. Disclosures No relevant conflicts of interest to declare.

FLT3-CD3 Bispecific Antibody Specifically Eliminates Normal Hematopoietic Progenitors and AML in Humanized Mouse Models

Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

Tumor Associated Macrophages Express High-Levels of FLT3 Ligand, Which Induces Activation of FLT3 Signaling That Promotes Survival of Neoplastic Cells in B-Cell Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare (&amp;lt;5%) in patients with B-ALL, suggesting an essential function of the ligand activating FLT3 in the initiation and progression of the disease. However, the presence, origin and function of FLT3 ligand (FLT3L) in B-ALL are not fully uncovered. In the present study, we observed high levels of FLT3L (234.7 ± 47.6 pg/ml) in plasma from 28 patients with B-ALL, compared with age-matched healthy donors (41.2 ± 15.5 pg/ml) by using ELISA. Among these samples, 12 were from patients achieved remission with negative minimal residue disease (MRD) but maintained high levels FLT3L (286.9 ± 71.2 pg/ml), suggesting cell-source(s) of FLT3L other than neoplastic B-ALL blasts. We hypothesized that tumor-associated macrophages (TAMs) from the microenvironment of B-ALL express FLT3L, because studies show that activated macrophages from patients with autoimmune disease express high levels of FLT3L to promote inflammation and destruction of local articular components. Previously, we cultured nurse-like cells, which are TAMs with a M2 phenotype from peripheral blood mononuclear cells (PBMCs) of patients with chronic lymphocytic leukemia (CLL) (Chen Y, et al. Blood. 2019; 134:1084-1094.). These cells are key components of microenvironment of CLL cells to support survival and migration of neoplastic B cells. In the present study, we cultured PBMCs from B-ALL patients and obtained non-neoplastic TAMs with a similar morphology to nurse-like cells. These cells were stained positive for CD68 and CD163, which are markers for M2 macrophages by immune fluorescent microscopy. We isolated RNA from purified B-ALL blasts or TAMs of the same patient for real-time PCR to measure the levels of FLT3L transcripts. FLT3L transcripts were abundant in the RNA isolated from TAMs, but were negligible in RNA isolated from B-ALL cells in each patient tested (N = 5). We also evaluated for secreted FLT3L protein via ELISA in the culture-medium of isolated TAMs and B-ALL cells with an equal cell number from 18 different patients. The cultured medium of TAMs had significantly higher concentration of FLT3L than B-ALL cells. Furthermore, we performed co-culture studies to assess the involvement of FL/FLT3 signaling in the survival of B-ALL cells cultured with TAMs. B-ALL cells co-cultured with TAMs had significantly more live cells detected by flow cytometry with annexin V and PI staining, compared with B-ALL cells cultured alone. Immunoblot showed that B-ALL cells co-cultured with TAMs had high levels of phosphorylated FLT3 and ERK, indicating the activated FLT3 signaling cascade in these cells. We speculate that the elevated level of FLT3L noted in the plasma of patients with B-ALL is produced primarily by TAMs, which reside in the microenvironment of neoplastic cells. Such high-level FLT3L may provide chemoresistance of B-ALL cells within the microenvironment. Treatment with FLT3 tyrosine kinase inhibitors may mitigate such effects, potentially enhancing the clearance of leukemia cells when used in combination with chemotherapy. Disclosures No relevant conflicts of interest to declare.

13q12.2 deletions in acute lymphoblastic leukemia lead to upregulation of FLT3 through enhancer hijacking

AbstractMutations in the FMS-like tyrosine kinase 3 (FLT3) gene in 13q12.2 are among the most common driver events in acute leukemia, leading to increased cell proliferation and survival through activation of the phosphatidylinositol 3-kinase/AKT-, RAS/MAPK-, and STAT5-signaling pathways. In this study, we examine the pathogenetic impact of somatic hemizygous 13q12.2 microdeletions in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) using 5 different patient cohorts (in total including 1418 cases). The 13q12.2 deletions occur immediately 5′ of FLT3 and involve the PAN3 locus. By detailed analysis of the 13q12.2 segment, we show that the deletions lead to loss of a topologically associating domain border and an enhancer of FLT3. This results in increased cis interactions between the FLT3 promoter and another enhancer located distally to the deletion breakpoints, with subsequent allele-specific upregulation of FLT3 expression, expected to lead to ligand-independent activation of the receptor and downstream signaling. The 13q12.2 deletions are highly enriched in the high-hyperdiploid BCP ALL subtype (frequency 3.9% vs 0.5% in other BCP ALL) and in cases that subsequently relapsed. Taken together, our study describes a novel mechanism of FLT3 involvement in leukemogenesis by upregulation via chromatin remodeling and enhancer hijacking. These data further emphasize the role of FLT3 as a driver gene in BCP ALL.

The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis.

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

High FLT3 Levels May Predict Sorafenib Benefit in Hepatocellular Carcinoma.

To identify a predictive biomarker of sorafenib for hepatocellular carcinoma personalized therapy. The patients treated with or without sorafenib after hepatocellular carcinoma recurrence from multicenters were matched with propensity score matching analysis. The expression levels of Fms-like tyrosine kinase 3 (FLT3) in hepatocellular carcinoma specimens of the matched patients (n = 276) were analyzed by IHC. The optimal cut-off point of FLT3 levels for overall survival (OS) was defined via Cutoff Finder. Subgroup analysis of OS was employed to investigate the association between FLT3 levels and sorafenib benefit. The predictive value was assessed via Cox regression models with an interaction term. Hepatocellular carcinoma and paratumoral normal tissues were used to investigate the expression and copy-number variation of FLT3. Patient-derived xenograft (PDX) models were used to confirm the association between FLT3 levels and sorafenib response. Patients with FLT3-high hepatocellular carcinoma exhibited a superior OS upon sorafenib treatment. High FLT3 levels were predictive of sorafenib benefit in terms of OS (P interaction = 0.00006). Copy-number losses and decreased expression of FLT3 in hepatocellular carcinoma were detected in about 64% of patients. Moreover, the PDXs derived from tumors with high FLT3 levels also displayed a better response to sorafenib. Sorafenib may be able to delay tumor progression in patients with FLT3-high hepatocellular carcinoma. This potential biomarker needs to be further validated in independent cohorts prior to helping stratify patients for precision therapy in advanced hepatocellular carcinoma.

Deficiency of core fucosylation activates cellular signaling dependent on FLT3 expression in a Ba/F3 cell system.

Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML.

DOCK2 Expression Affects Leukemogenesis and Disease Progression in a Murine Model of FLT3/ITD Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and an internal tandem duplication (ITD) mutation portends a poor prognosis. FLT3/ITD activates Rac1 GTPase, and both proteins play key roles in DNA damage response (DDR). Disease progression in FLT3/ITD AML is often heralded by copy-neutral loss of heterozygosity (CN-LOH) at the FLT3 locus, which is typically caused by duplication of the mutant allele through homologous recombination with loss of the wild-type (WT) allele. We previously identified dedicator of cytokinesis 2 (DOCK2) as a FLT3/ITD-interacting protein. The DOCK family of proteins acts as guanine nucleotide exchange factors for the Rho family of GTPases, including Rac1. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts in FLT3/ITD AML. Knockdown of DOCK2 in FLT3/ITD cell lines results in reduced Rac1 activity, decreased expression and activity of FLT3 and DDR factors, and increased sensitivity to cytarabine. In this study, we used mouse models to further investigate the relationship between DOCK2 and FLT3/ITD. Mice with a Dock2 mutation (Dock2/Hsd) that results in a marked reduction in Dock2 expression were bred with mice that carry a Flt3/ITD mutation (Flt3+/ITD). Quantitative PCR of mouse bone marrow (BM) revealed that homozygous expression of Dock2/Hsd markedly decreased the expression of Flt3 and key DDR factors in both WT and Flt3+/ITD mice. Flow cytometric analysis of the BM demonstrated that Dock2/Hsd resulted in partial reversal of the myeloproliferative neoplasm that develops in Flt3+/ITD mice, with a significant reduction in multipotent progenitors (MPP) in Flt3+/ITD;Dock2Hsd/Hsd (0.026%) vs. Flt3+/ITD mice (0.075%; P=0.048) at 3 months. To assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine model of FLT3/ITD acute leukemia that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. It was previously shown that Flt3+/ITD;NHD13 mice develop acute leukemia with 100% penetrance and short latency. We found that the median survival of Flt3+/ITD;NHD13 mice (117 days) was not significantly affected by heterozygous expression of Dock2/Hsd (123 days); however, the median survival of Flt3+/ITD; NHD13; Dock2Hsd/Hsd mice was extended to 198 days (P=0.001). In contrast, Dock2/Hsd provided no survival benefit to mice that carried either Flt3+/ITD or NHD13 alone. The prolonged survival of Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice was accompanied by delayed onset of LOH at the Flt3/ITD locus. At 3 months, approximately half of Flt3+/ITD;NHD13;Dock2+/+ (47%; n=19) and Flt3+/ITD;NHD13;Dock2+/Hsd (57%; n=14) mice exhibited LOH in the peripheral blood, whereas only 11% of Flt3+/ITD; NHD13;Dock2Hsd/Hsd mice showed LOH (n=18; P=0.008 vs. Dock2+/Hsd and P=0.029 vs. Dock2+/+). Most of the moribund Flt3+/ITD;NHD13 mice (n=13) developed AML with minimal maturation (70%), and the remainder developed T-lymphoblastic leukemia with an early T-precursor phenotype (ETP-ALL). In contrast, moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice (n=14) exhibited a more diverse disease spectrum: 30% mixed phenotype acute leukemia, 21% AML with minimal maturation, 21% ETP-ALL, 21% AML with maturation, and 7% acute erythroid leukemia (AEL). All moribund Flt3+/ITD;NHD13 mice showed complete LOH in BM; however, only 68% of moribund Flt3+/ITD;NHD13;Dock2Hsd/Hsd mice exhibited complete LOH, with the rest showing partial or no LOH. Notably, all mice that exhibited partial or no LOH developed AEL or AML with maturation, diseases that were present in NHD13 mice but were not observed in Flt3+/ITD;NHD13 mice. This suggests that suppression of Dock2 mitigates the effects of Flt3/ITD, allowing the NHD13 mutation to drive the development of disease. These findings demonstrate that decreased DOCK2/Rac1 activity delays LOH at the FLT3 locus and disease progression in a murine model of FLT3/ITD leukemia. Thus, pharmacologic inhibition of the Rac1 signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Disclosures Small: Pharos I, B &amp; T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees. Duffield:Boston Biomedical/Sumitomo Dainippon Pharma Co., Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; MedImmune: Consultancy.