ALBERT

Description: Gilteritinib and Venetoclax synergize to eliminate FLT3/ITD+ leukemia cells through BIM Abstract Acute myeloid leukemia (AML) is characterized by a clonal proliferation of immature myeloid cells in the bone marrow or other tissues. The most commonly mutated gene in AML is FMS-like tyrosine kinase (FLT3). FLT3 downstream signaling pathways include PI3K/AKT, STAT5 and MAPK, which affect apoptosis, differentiation and cell proliferation. An internal tandem duplication mutation in FLT3 (FLT3/ITD) is identified in approximately 25% of patients with AML, and this mutation is associated with a particularly poor prognosis. A subset of AML also has a point mutation in the tyrosine kinase domain of FLT3 (FLT3/TKD); however, this mutation does not have the same pronounced impact on prognosis as the FLT3/ITD mutation. Since tyrosine kinases are an attractive drug target, tyrosine kinase inhibitors (TKIs) that target FLT3 have been developed, including recent agents that show enhanced specificity, such as Gilteritinib. Despite these advances, TKI monotherapy continues to show limited success, indicating that combination therapy is likely necessary for the effective treatment of FLT3/ITD AML. As FLT3 signaling pathway activity is known to be anti-apoptotic, in this study we investigated the combinatorial effect of a FLT3-selective TKI and the BCL-2 inhibitor Venetoclax. The BCL-2 protein plays a key role in apoptosis, with anti-apoptotic/prosurvival effects. Venetoclax is a selective BCL-2 inhibitor, and is used clinically in the treatment of chronic lymphocytic leukemia and relapsed/refractory AML. We first investigated the combinatorial effect of treatment with Gilteritinb and Venetoclax in a FLT3/ITD+ leukemia cell line (Molm14). Combined treatment with Gilteritinib (20nM) and Venetoclax (80nM) reduced cell proliferation by 83.7%, as compared to Gilteritinib (62.2%, P

Description: Introduction: The MPN, polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) are clonal stem cell disorders, which share mutations constitutively activating the physiologic signal transduction pathways for hematopoiesis. Although the MPN have different natural histories, they share in common transformation to myelofibrosis and acute leukemia at differing frequencies and not explainable by a specific mutation. Impaired MPL expression, due to incomplete glycosylation is another common denominator amongst the MPN. Significantly, MPL is the only hematopoietic growth factor receptor expressed in MPN HSC, and we have demonstrated that the PV phenotype in a JAK2 V617F transgenic (V617tg) mouse could be abrogated by elimination of MPL or its ligand, TPO. Impaired MPL expression in the MPN cannot be completely explained by receptor-activated down regulation because not all MPN driver mutations directly activate MPL. However, we have discovered another common denominator in the MPN, variant MPL splicing, eliminating 7 amino acids in the MPL N-terminal domain, a common hotspot for both MPL driver and inactivating mutations, which impairs MPL glycosylation and expression. Methods: To determine the role of the MPL splice variant (MPL SV) in MPN pathophysiology, we cloned the full length MPLSV cDNA and created a transgenic mouse (MPLSV tg) using the same VAV promoter as the V617Ftg mouse to ensure hematopoietic cell-specific transcription. The MPLSV tg mice were produced by pronuclear injection of purified MPLSV cDNA into B6SJLF1 mice. Founders were crossed into a C57Bl/6 background, and then were crossed with V617Ftg mice in either an MPL knockout or wild type background. Mice were phenotyped by blood counts and necropsy with morphologic and immunophenotyping of bone marrow and tumor masses. Results: Amongst 19 founder B6SJLF1 mice, 6 expressed the MPLSV transgene with copy numbers ranging from one to 30; single copy number mice had no hematopoietic phenotype but all mice with higher MPLSV tg copy numbers had thrombocytopenia (median platelet count 500,000/µL; range 200- 600,000; wild type, 750,000/µL; 550-950,000). MPLSV tg mice from several different founders bred into a C57Bl/6 background for 6 generations maintained consistent copy numbers, Mendelian ratios and hematopoietic phenotypes with 100% penetrance and an inverse correlation between MPLSV copy number and the platelet count. The number of double transgenic (V617Ftg/MPLSV tg) offspring observed in the MPLSV tg to V617Ftg crosses were slightly lower than expected (20% vs 25%), indicating reduced embryo viability. Four week old V617Ftg/MPLSV tg mice displayed tumorous abnormalities of the head and hips as well as small size and failure to thrive (median weight 8.5 gms; range 8-9; wild type, 13 gms; 10-14), and enlarged spleens (median weight 0.29 gms; range 0.15-0.64; wild type 0.086 gms; 0.05-0.10). Histologically, the V617Ftg/MPLSV tg-associated head and hip abnormalities represented monomorphic myeloid sarcomas extending through the calvarium both extra and intracranially and from the femur into surrounding muscle. Both the marrow and spleen were diffusely infiltrated by large blasts with abundant basophilic cytoplasm, expressing CD34, CD61 and CD117 but lacking CD127, consistent with a myeloid origin. All V617Ftg/MPLSV tg mice either died or required humane sacrifice by 6 weeks. The extramedullary tumor was transplantable in secondary recipients, and flow cytometry-based phenotyping showed that the tumors (both primary and secondary) were CD34, CD117, CD61 and CD71- positive. Penetrance of the leukemia phenotype was 100% in V617Ftg/MPLSV tg from all founder lines with multiple copies of the MPLSV tg, whether in an MPL knockout or wild type background. The leukemia phenotype was never observed with the MPLSV tg alone or with V617Ftg alone despite observation of over 300 V617Ftg mice up to 50 weeks of age. Conclusion: We identified an MPL SV in human MPN that was functional in mice with a dominant-negative effect with respect to platelet production and at the same time synergized with JAK2 V617F to create a fulminant myeloid malignancy. We have recently shown that knockout of MPL or TPO alone abrogates the PV phenotype in the V617Ftg mouse, whereas the MPLSV uniquely drives a highly penetrant and fulminant leukemia, establishing MPL and TPO as targets for mitigation of malignant transformation in the MPN. Disclosures Spivak: Incyte: Membership on an entity's Board of Directors or advisory committees. Moliterno:incyte: Membership on an entity's Board of Directors or advisory committees.

Description: Background: WT1 is a zinc finger transcriptional regulator and acts as a tumor suppressor gene in various cell types. WT1 mutations are reported in approximately 10% of both adult and pediatric patients with acute myeloid leukemia (AML), and at a lower frequency in patients with myelodysplastic syndome (MDS). Reported mutations consist of insertions, deletions or point mutations, and are thought to alter WT1 DNA-binding ability and result in a loss of function. WT1 mutations are associated with FLT3/ITD mutations in AML, suggesting possible leukemogenic cooperativity, and yet WT1 mutations have been independently associated with treatment failure and a poor prognosis. Recently, a physical interaction demonstrated between WT1 and TET2 suggests a common functional pathway, and explains the mutual exclusivity of these mutations in AML. Despite these observations, the functional contribution of WT1 mutations in hematologic malignancies is not entirely understood. To our knowledge, we are the first to describe here a hematologic phenotype in a WT1 mutant mouse model and in a novel WT1 mutant x FLT3/ITD crossbred mouse model. Methods: Knock-in WT1 mutant mice are heterozygous for missense mutation R394W in the DNA-binding domain, which has been described in cases of human AML. Mice with a heterozygous 18-bp ITD knocked into the FLT3 gene were crossbred with the WT1 mutant mice, and Kaplan-Meier survival analysis was performed across genotypes. CBCs and BM cytospin morphology from moribund mutant mice were compared to wild type controls. To create a transplant model, 2e6 whole BM cells from each genotype were injected into lethally irradiated congenic mice. Competitive transplants were performed by injecting a 1:1 ratio of CD45.1 wild type (control) cells with CD45.2 WT1 mutant or wild type (test) cells into lethally irradiated C45.1 recipients. Results: We noted an expansion of lineage negative cells and various progenitor cell compartments in WT1 mutant (WT1mut) BM relative to wild type (wt); including the megakaryocyte-erythroid progenitor (MEP) compartment. WT1mut BM cells from two-month old mice showed an increased ability to serially replate in methylcellulose culture compared to wt BM cells, demonstrating aberrantly enhanced self-renewal capacity. WT1mut mice demonstrated a trend towards an inferior late survival compared to wt in survival analysis, and several moribund WT1mut mice were found to have anemia and erythrodysplasia. Most ITD mice developed a fatal myeloproliferative neoplasm (MPN), as previously described. Interestingly, double mutant mice (WT1mut+ITD) had an inferior survival compared to ITD (p

Description: Introduction: While immunosuppression for solid organ transplant is associated with an increased risk of lymphoproliferative disease (LPD), this has been more difficult to establish in autoimmune disorders, even though patients are often treated with similar agents. One reason is that autoimmune disease may elevate baseline LPD risk. However, associations have been shown with certain rare types of LPD; most strikingly hepatosplenic lymphoma, now known to occur as a consequence of anti-TNF-alpha therapy in young men with inflammatory bowel disease (IBD) (J Ped Gast Nutr. 2007; 44:265-7). We have noticed a rise in the incidence of another rare lymphoma in autoimmune disease patients: primary central nervous system (PCNS) LPD. Six cases have been diagnosed at our institution since 2010, with none before that dating back to onset of electronic records in 1986. All of these patients were taking mycophenolate mofetil (MMF) and/or thiopurines. A similar rise in reported cases has been seen in the literature (Fig 1) with suggestion of but no direct association with drug treatment shown. We systematically investigated this trend. Methods: We searched our pathology database to identify all LPD cases diagnosed over a 28-year period in patients treated for autoimmune disease as well as all similar cases involving the CNS reported in the literature over the past 40 years. Statistical analyses were performed using the Fisher's exact test. Results: We identified 44 cases of LPD arising in patients treated for autoimmune disease, including 6 with PCNS disease (Table 1). Of LPDs in patients on anti-TNF-alpha agents, 4/5 had a T-cell phenotype, and 3 had IBD. By contrast, in patients who developed LPD while taking methotrexate, the majority for rheumatoid arthritis, only 1/18 had a T-cell phenotype. Instead they were categorized as polymorphous, Hodgkin or large B-cell morphologies, which were frequently EBV-positive (67%), but never involved the brain (0/18). The LPDs arising in patients on MMF and/or thiopurines showed a similar morphologic profile but were more likely to involve the CNS. In particular, MMF was significantly associated with PCNS compared to non-CNS disease (p

Description: Background: Burkitt lymphoma typically involves the bone marrow in advanced stage disease. Pure leukemic presentation of Burkitt lymphoma (pure Burkitt leukemia) with negative radiographic findings is uncommon but considered clinically as stage IV disease. The WHO classification clearly distinguishes Burkitt leukemia (formerly classified as FAB-L3) from B lymphoblastic leukemia, as the former has a mature B-cell phenotype (expresses surface immunoglobulin) and characteristically harbors a MYC translocation with an immunoglobulin partner. There is limited prognostic information on patients with pure Burkitt leukemia (PBL) because they are typically combined with Burkitt lymphoma patients with widespread disease (WBL). Methods: We performed a multicenter retrospective analysis of newly-diagnosed Burkitt lymphoma cases involving the bone marrow at four academic medical centers from 2000-2013. Inclusion criteria included availability of clinical information and follow-up data, MYC rearrangement, and treatment with intensive chemotherapy (Hyper-CVAD or ALL-like regimens). Pediatric patients were excluded (age 〈 21 years). Diagnosis was established by the institutions' expert hematopathologist in conjunction with cytogenetic, flow cytometric, and radiographic findings. Patients were placed in the pure Burkitt leukemia (PBL) group if they had bone marrow involvement but were otherwise radiographically negative (i.e. PET negative). Kaplan-Meier analysis was used to examine the difference in overall survival between the WBL and PBL groups. We also determined prognostic factors associated with survival in univariate and multivariate Cox regression analyses. Results: We identified 51 patients with bone marrow involvement by Burkitt lymphoma. Of these patients, 24 were excluded because of a lack of clinical or follow-up information (15), pediatric age group (7), no treatment initiated (1), or misdiagnosed and given inadequate treatment (1). Of the 27 patients that met our inclusion criteria, 11 patients had pure Burkitt leukemia (PBL) and 16 had tissue involvement with bone marrow disease (WBL). The male-to-female ratio in the PBL group was 4.5:1 and in the WBL 4:1 (p=0.23). The median age for the PBL was 46 years vs 47 years in the WBL (p=0.34). CSF involvement was similar in both groups (30% PBL vs. 38.5% WBL; p=0.14); with elevated LDH levels in all patients in both groups (PBL median 3399 IU/L, WBL 2550 IU/L; p=0.81). In general, the WBL group had more complex karyotypes and a significantly greater number of cases with chromosome 1q abnormalities (5/7, 71%) compared to the PBL group (1/5, 20%). In the survival analysis, patients with PBL exhibited significantly better survival with a 5-year-overall survival of 79.5% (95% CI: 57.7-100%) vs. 18.4% (95% CI: 5.3-63.3%) in the WBL group (p=0.002). Conclusion: To the best of our knowledge, this is the largest series comparing adults with pure Burkitt leukemia and Burkitt lymphoma with widespread disease. Within the limitations of a retrospective analysis, the 5-year overall survival for the widespread Burkitt lymphoma (WBL) group is inferior compared to pure Burkitt leukemia (PBL) treated with intensive chemotherapy. Interestingly, the WBL group had more complex karyotypes compared to the PBL group. Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Disclosures No relevant conflicts of interest to declare.

Description: Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3-ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3-ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse harboring an internal tandem duplication at the endogenous Flt3 locus, that develops a fatal myeloproliferative neoplasm (MPN), but fails to develop full blown leukemia, suggesting additional mutations are necessary for transformation. Global genomic sequencing studies have identified a substantial subset of patients in which FLT3-ITD and DNMT3A mutations are concomitantly present. Moreover, the co-occurrence of these mutations is significantly associated with adverse clinical outcomes (Patel JP, et al. NEJM. 2012;366:1079-89). Based on these observations, we investigated the potential cooperativity of FLT3-ITD and mutant DNMT3A to drive leukemogenesis using genetically engineered mouse strains. In accordance with mounting evidence that DNMT3A mutations result in a loss of function, we used a mouse model harboring floxed Dnmt3a alleles (Dnmt3af/f), and a lymphocyte specific Cre transgene (Mx1-Cre+), which is activated upon injection with Polyinosinic-polycytidylic acid (PiPC). We used a substrain of our Flt3-ITD knock-in mice, which retains a floxed Neomycin (Neo) selection cassette from the initial targeting (Flt3ITDneo/+), and reduces expression of the mutant allele until PiPC treatment. Mice were bred to contain both types of mutation and were injected with PiPC intraperitoneally at 8 weeks of age and monitored for disease development. Mice containing both the FLT3/ITD and Dnmt3af/f developed leukemias. Hempatopoietic tissues were examined morphologically and by flow cytometry. Interestingly, deletion of Dnmt3a significantly reduced median survival of Flt3-ITD mice in a dose-dependent manner, with median survival of 162 days and 260 days for Dnmt3af/f;Flt3ITDneo/+ and Dnmt3af/+;Flt3ITDneo/+, respectively (Figure 1). Both genotypes confer a significantly shorter survival time compared to Flt3ITDneo/+ alone controls, which have a median survival of 412 days. Moribund mice exhibited elevated white blood cell counts and splenomegaly, and developed a spectrum of diseases. As expected, Flt3ITDneo/+ mice solely developed MPN, while Dnmt3af/f;Flt3ITDneo/+ and Dnmt3af/+;Flt3ITDneo/+ developed a spectrum of neoplasms including MPN, T-ALL, and AML. Dnmt3a dosage influences the disease phenotype, as Dnmt3af/f;Flt3ITDneo/+ (n=18) develop T-ALL (46%), AML (31%), and MPN (23%), while Dnmt3af/+;Flt3ITDneo/+ mice (n=17) present with T-ALL (14%), T cell Lymphoma (14%), AML (43%), and MPN (29%). Recent work has demonstrated that Dnmt3a deletion in hematopoietic stem cells (HSC) promotes self renewal and expansion of the LT-HSC pool (Challan GA, et al. Nat Genet. 2011;44:23-31). Conversely, Flt3-ITD disrupts LT-HSC quiescence, resulting in depletion of this compartment (Heiser D & Chu SH, et al. Cell Stem Cell. 2012;11:346-58). To investigate if Dnmt3a deletion might restore or expand the LT-HSC compartment when combined with Flt3-ITD, we examined the bone marrow of mice 8 weeks post PiPC injection, and found that Dnmt3af/f;Flt3ITDneo/+ mice displayed an expansion of the LT-HSCs, ST-HSCs, and Multipotent Progenitors compared to wild type, Dnmt3af/f;Flt3+/+, and Flt3-ITD mice. The HSC populations of Dnmt3af/+;Flt3ITDneo/+ mice exhibit similar proportions compared with Flt3-ITD mice, with a modest increase in the MPP population. These results illustrate, for the first time, that Dnmt3a loss cooperates with Flt3-ITD to generate myriad hematopoietic neoplasms, including AML. In combination with Flt3-ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and Dnmt3a mutations in AML, as well as subsets of T-ALL, suggests the Dnmt3af/f;Flt3ITDneo/+ model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts. Figure 1. Kaplan-Meier Survival Curve. Median survival: Dnmt3af/f;Flt3ITDneo/+ = 162 days (n=24), Dnmt3af/+;Flt3ITDneo/+ =260 days (n=20), Flt3ITDneo/+ = 412 days(n=12). Figure 1. Kaplan-Meier Survival Curve. Median survival: Dnmt3af/f;Flt3ITDneo/+ = 162 days (n=24), Dnmt3af/+;Flt3ITDneo/+ =260 days (n=20), Flt3ITDneo/+ = 412 days(n=12). Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. While most relapses occur within 2 years, a small proportion of patients present with late relapse (LR) after 5 years. As there are very few studies addressing the pathobiology of LR-DLBCL, the aim of this study is to further characterize the clinical, pathologic and molecular features of these neoplasms. METHODS: A retrospective analysis of all patients with DLBCL treated at Johns Hopkins Hospital between 1984 and 2013 was performed. Patients with low-grade lymphoma at any time-point were excluded. Disease-free intervals (DFI) of 5 years or greater were designated as LR. Five paired diagnostic (D) and relapse (R) samples were available for further studies. DNA was extracted from formalin fixed paraffin embedded tissue. IGH gene rearrangement status was determined by PCR. SNP microarray was performed, and copy number variations (CNV) were defined as loss or gain of signal over at least 2 megabases. Targeted next generation sequencing (NGS) using a cancer hotspot panel was also performed. Variant calls were generated using Torrent variant caller and a laboratory-developed analysis pipeline. RESULTS: One hundred thirty-three patients with relapsed DLBCL were identified. Forty-three (32.3%) patients were diagnosed in the pre-rituximab era. One hundred fourteen (85.7%) patients had early relapse (ER) with 99 (74.4%) patients recurring within 2 years. Nineteen (14.3%) patients had LR (mean 7.9 years; median 7.3 years; up to 15.6 years). There were no significant differences in age at diagnosis, race, staging marrow status, or overall survival (OS) in ER versus LR patients. Extra-nodal presentation at diagnosis (89.5% vs. 65.8%; p = 0.04) and extra-nodal-only disease over time (73.7% vs. 48.2%; p = 0.04) were more common in LR cases. Both groups had similar rates of recurring at a different site from the original disease (79.3% vs. 89.5%; p = 0.30). Table 1. Molecular profile of paired D and R DLBCL Patient IGH clonality comparison (D vs. R) Clonal Heterogeneity (D / R) Total CNVs (D / R) Shared CNVs Unique CNVs (% of D / % of R) 1 Same + / + 24 / 21 15 37.5 / 28.6 2 Same + / + 15 / 32 11 26.7 / 65.6 3 Same + / + 32 / 15 7 78.1 / 53.3 4 2 in D / 1 persists in R + / + 8 / 20 1 87.5 / 95.0 5 Different - / + 4 / 5 0 100 / 100 The average DFI was 7.1 years in the 5 LR patients selected for additional studies. IGH gene rearrangement analysis demonstrated identical D and R IGH clones in 3 cases (Table 1). Patient 4 showed 2 rearranged alleles at D with only 1 persisting at R. Patient 5 had lymphomas with unique IGH rearrangements. SNP microarray data demonstrated the presence of clonal heterogeneity in all but 1 sample (4 of 5 at D; 5 of 5 at R). Among the 4 patients with clonally related IGH gene rearrangements, there was only partial overlap in CNVs (approximately 40% on average) between the D and R lymphomas. The average CNVs was similar in the D and R samples (16.6 vs. 18.6 respectively; p = 0.75). Chromosomes 2, 3, 6, 9, and 17 were frequently altered, and CNVs involving the BCL-6, CDKN2A, TP53, and MYC loci were also commonly seen; but there was no systematic difference between the CNVs identified at D and R. NGS showed a variety of mutations, but no consistent pattern of mutations acquired at R. There was a nonsense mutation in exon 2 of CDKN2A in the R sample in patient 1, and both D and R samples showed the same copy-neutral loss-of-heterozygosity of 9p encompassing the CDKN2A gene. In addition, missense mutations of TP53 were detected in patients 4 (only at R) and 5 (only at D). CONCLUSIONS: This study demonstrates that LR-DLBCL is an uncommon phenomenon with most cases representing recurrence of the original disease. LR patients have similar OS as ER patients, and the only clinical factors segregating LR from ER are higher rates of extra-nodal presentation and extra-nodal-only sites of disease. Although most paired D and R cases share IGH clones, there is clear evidence of clonal heterogeneity with clonal evolution over time. This suggests that DLBCL may contain minor subclones not susceptible to chemotherapy, which persist subclinically acquiring additional mutations over time eventually generating clinically-evident relapse. In rare cases, the late “relapse” may occur as an unrelated lymphoma that arises spontaneously or secondary to the mutagenic effects of chemotherapy. The precise mechanism of this long latency is yet unclear, and requires further investigation. Disclosures Borowitz: Becton Dickinson Biosciences: Research Funding.

Description: Short telomeres have been linked to cancer risk, yet other evidence supports them being tumor suppressive. Here, we report cancer outcomes in individuals with germline mutations in telomerase and other telomere-maintenance genes. Among 180 individuals evaluated in a hospital-based setting, 12.8% had cancer. Solid tumors were rare (2.8%); nearly all were young male DKC1 mutation carriers, and they were generally resectable with good short-term outcomes. Myelodysplastic syndrome (MDS) was most common, followed by acute myeloid leukemia (AML); they accounted for 75% of cancers. Age over 50 years was the biggest risk factor, and MDS/AML usually manifested with marrow hypoplasia and monosomy 7, but the somatic mutation landscape was indistinct from unselected patients. One- and 2-year survival were 61% and 39%, respectively, and two-thirds of MDS/AML patients died of pulmonary fibrosis and/or hepatopulmonary syndrome. In one-half of the cases, MDS/AML patients showed a recurrent peripheral blood pattern of acquired, granulocyte-specific telomere shortening. This attrition was absent in age-matched mutation carriers who did not have MDS/AML. We tested whether adult short telomere patients without MDS/AML also had evidence of clonal hematopoiesis of indeterminate potential–related mutations and found that 30% were affected. These patients also primarily suffered morbidity from pulmonary fibrosis during follow-up. Our data show that the Mendelian short telomere syndromes are associated with a relatively narrow cancer spectrum, primarily MDS and AML. They suggest that short telomere length is sufficient to drive premature age-related clonal hematopoiesis in these inherited disorders.

Description: The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying FLT3/ITD mutations have a poor prognosis. Despite continuing progress in the development of more effective FLT3 inhibitors, long-term success in inhibition of FLT3 activity in AML patients is still elusive. In order to achieve a better understanding of FLT3 biology and more effective strategies for the inhibition of FLT3 activity, a screen was performed on leukemia cell lines to search for FLT3-interacting proteins. One of the proteins identified in the screen was dedicator of cytokinesis 2 (DOCK2). The DOCK family of proteins acts as guanine nucleotide exchange factors (GEFs) for Rho family of GTPases, which includes Rac GTPases. DOCK2 expression is limited to hematopoietic cells, and is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells, and cell-cell adhesion and bone marrow homing of various immune cells. We first verified that DOCK2 is expressed in primary AML samples from patients, and co-immunoprecipitation experiments showed that DOCK2 interacts with both wild-type FLT3 and FTL3/ITD in these cells. Co-immunoprecipitation experiments using leukemia cell lines demonstrated that DOCK2 interacts with FLT3, FLT3/ITD, FLT3/D835Y and FLT3/D835H, and that it predominantly interacts with the unphosphorylated form of FLT3. Knock-down of DOCK2 by shRNA did not significantly affect the growth of cell lines that lack expression of FLT3, but greatly reduced growth of cell lines expressing amplified wild type FLT3 (Sem K2), FLT3/D835H (HB11;19) and FLT3/ITD (MV4;11). Accordingly, colony formation assays revealed that cell lines with elevated expression of wild type or mutant FLT3 produced fewer, smaller and more compact colonies when the expression of DOCK2 was decreased, while colonies from cell lines lacking FLT3 expression showed no significant difference in response to the knock-down of DOCK2. Furthermore, an Annexin V binding assay indicated that reduction in DOCK2 expression level greatly sensitized cells with elevated FLT3 activity (MV4;11 and Sem K2) to cytarabine, resulting in increased apoptosis, but no significant sensitization was observed in cell lines that lack FLT3 expression. These findings demonstrate that DOCK2 interacts with FLT3 in leukemia cell lines, and suggest that this interaction has important roles in regulating the survival of leukemia cells with elevated FLT3 activity, both alone and in combination with conventional anti-leukemic agents. Therefore, DOCK2 is a potential therapeutic target for AML treatment, and better understanding of the interaction between DOCK2 and FLT3 may contribute to the development of novel strategies to effectively inhibit FLT3 activity in AML patients. Disclosures No relevant conflicts of interest to declare.