FLT3 Signaling Research Articles

Abstract 834: FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation

Abstract Background: In 30% of acute myeloid leukemia (AML) patients, the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) causes constitutive FLT3 signaling, and these patients relapse rapidly following response to chemotherapy. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to its proliferative and antiapoptotic effects and also phosphorylates and stabilizes it in a positive feedback loop. FLT3 activation also inhibits the tumor-suppressor protein phosphatase 2A (PP2A), causing stabilization of Pim-1 protein. FLT3 inhibitors and PP2A-activating drugs (PADs) are known to synergistically induce cytotoxicity in cells with FLT3-ITD, and here we studied mechanisms underlying this effect. Methods: Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 cells, with FLT3-ITD, were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and the PAD fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Apoptosis was measured by Annexin V/propidium iodide staining. Drug combination indexes were determined by the Chou-Talalay method with CompuSyn software. Pim-1 kinase, Myc, MycSer62, MycThr58, STAT5, phospho-STAT5, PP2A and phospho-PP2A levels were measured by immunoblotting. Cycloheximide was used to assess protein stability. Results: Concurrent treatment with ASP2215 or AC220 and FTY720 decreased growth and enhanced apoptosis of cells with FLT3-ITD, relative to single drugs, and produced synergistic cytotoxicity. Concurrent treatment sequentially decreased expression of the 44 kDa Pim-1 isoform (Pim-1L) and then c-Myc, via decreased Pim-1L and c-Myc protein stability. Co-incubation with the proteasome inhibitor MG-132 prevented drug-induced degradation and restored Pim-1L and Myc expression in cells co-treated with FLT3 inhibitor and PAD. Drug-induced Myc destabilization/proteasomal degradation was associated with decreased MycSer62 and increased MycThr58 levels. Treatment with the pan-Pim kinase inhibitor AZD1208 prior to ASP2215 or AC220 and FTY720 treatment markedly increased MycThr58 levels but did not affect MycSer62, consistent with both Pim-dependent and -independent effects of FLT3 inhibition and PP2A activation on c-Myc expression. Conclusions: Combined FLT3 inhibitor and PAD treatment synergistically induces cytotoxicity in cells with FLT3-ITD by enhancing Pim-1 and Myc proteasomal degradation, and effect on Myc is both Pim-1-dependent and -independent. The data support in vivo testing of FLT3 inhibitor and PAD combinations. Citation Format: Mario Scarpa, Shivani Kapoor, Sikemi Ibikunle, Andre Rush, Patrick R. Baldwin, Danilo Perrotti, Maria R. Baer. FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 834.

Abstract 2559: Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia

Abstract Background: FLT3 (CD135) is a receptor tyrosine kinase expressed by hematopoietic progenitor cells, and when activated by ligand binding, FLT3 signaling can induce survival, proliferation and differentiation. Acute myeloid leukemia (AML), an indication with high unmet need, derives from hematopoietic progenitor lineage cells; consequently, many AML patient samples retain FLT3 expression on circulating leukemic cells. In comparison to the wide expression seen in AML patient samples, normal tissue expression of FLT3 is restricted to the hematopoietic compartment: a subset of hematopoietic stem cells, a subset of lineage committed progenitor cells and dendritic cells. Cell surface FLT3 protein was not detectable in those solid tissues that express quantifiable FLT3 mRNA (cerebellum, pancreas). As a result of this favorable expression profile, targeting AML cells using T cells expressing a chimeric antigen receptor (CAR) directed against FLT3, is expected to provide benefit to patients. Methods: A panel of fully-human, anti-human FLT3 antibodies were generated, screened for specific FLT3 binding, converted to single chain variable fragments (scFv) and re-evaluated for binding and stability. Three anti-FLT3 scFv sequences were selected for incorporation into CAR constructs. T cells transduced with these CAR constructs were evaluated in vitro for cytotoxicity, proliferation and cytokine secretion, and in vivo in a mouse xenograft model. The lead anti-FLT3 scFv was combined with an anti-CD3 scFv (bispecific T cell engager (BiTE®) format) for evaluation in non-human primates (NHP). Results: Human T cells engineered to express anti-FLT3 CAR constructs were evaluated for cytotoxicity, proliferation and cytokine secretion in the presence of three FLT3-positive cell lines, each with a different number of surface-expressed FLT3 receptors, and one FLT3-negative cell line. While all constructs were selectively active against FLT3-positive cells, one construct was more active than the others against cells expressing very low levels of FLT3; 90% depletion of target cells with ~1600 receptors/cell was observed after 40 hours of co-culture with CAR T cells in a 1:1 ratio. This lead construct was further evaluated in a mouse xenograft model, where FLT3 CAR-T cells provided a survival advantage. The anti-FLT3 scFv used in the lead CAR construct was evaluated in NHP in the form of a BiTE®. In this 16-day, multi-dose study, the FLT3 BiTE® induced elimination of FLT3+ cells as assessed by depletion of FLT3 mRNA-expressing cells (97% reduction in FLT3 mRNA) in the circulation and increased levels of soluble FLT3 ligand in serum. Conclusions: These results demonstrate the potential of using FLT3 CAR-T cell therapy for the treatment of AML. Citation Format: Tara L. Arvedson, Alice Bakker, Herve Lebrec, Gregor Adams, Armen Madiros, Priya Koppikar, Mercedesz Balazs, Mei Gong, Yan Zheng, Rebecca Goldstein, Tony Polverino, Lawren Wu, Angela Coxon. Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2559.

Abstract 794: CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, targets multiple pathways to kill diverse subtypes of acute myeloid leukemia and B-cell malignancy in vitro

Abstract Small molecules targeting the FLT3 kinase with an internal tandem duplication mutation (FLT3-ITD) and the Bruton's Tyrosine Kinase (BTK) have shown great potential to treat hematologic malignancies. However, acquired mutations have emerged in resistant/relapsed patients treated with the FLT3 inhibitor quizartinib (Smith et al., 2012) or the BTK inhibitor ibrutinib (Woyach et al., 2017), respectively. This study explored the potency and molecular mechanisms of CG'806, a pan-FLT3/pan-BTK inhibitor, in hematologic malignancies relative to other FLT3 or BTK inhibitors commercialized or in development. In FLT3-ITD AML cells, CG'806 induced apoptosis through inhibition of FLT3 signaling (decreased phospho-FLT3, -STAT5 and -ERK) and promotion of G0/G1 cell cycle arrest determined by immunoblotting and flow cytometry, and CG'806 was approximately 10-fold more potent than quizartinib. Although FLT3-ITD is found in 25-30% of AML patients, most AML patients express wild type (WT) FLT3. CG'806 had an IC50 = 11 nM against FLT3 WT-transfected Ba/F3 cells and was superior to quizartinib, gilteritinib and crenolanib FLT3 inhibitors (1,956, 500 and 2,617 nM, respectively). In FLT3-WT AML cell lines, or Ba/F3 cells transfected with FLT3-WT, D835Y, ITD+D835Y, or ITD+F691L, CG'806 markedly decreased phosphorylation of BTK, aurora kinases (AURK) and H3S10, resulting in G2/M arrest or polyploidy, and apoptosis with less or no effect on FLT3-WT activity. In contrast, quizartinib did not affect BTK or AURK signaling, and CG'806 was >2,000-fold more effective than quizartinib on FLT3-WT AML cells in MTS based cell proliferation assays. In B cell malignancies, BTK signaling plays a pivotal pathogenic role. CG'806 decreased BTK phosphorylation in all malignant B cell lines tested (n =10) and inhibited cell proliferation and colony formation 50-6,000 times more potently than ibrutinib, an effect which could not be explained by the exclusive inhibition of BTK signaling. Further analysis revealed CG'806 effectively inhibited AURK signaling and caused polyploidy and apoptosis in B cell lines, sequelae which were not induced by ibrutinib. BTK-C481S is the most common mutation induced by treatment with ibrutinib and is associated with ibrutinib resistance in the clinic. CG'806 equivalently inhibited BTK-WT and BTK-C481S in HEK293 transfected cells, whereas ibrutinib was much less potent against the BTK-C481S mutant. The ability of CG'806 to target all WT and mutant forms of FLT3 and BTK, and to inhibit multiple signaling pathways, produces killing of diverse subtypes of hematologic malignancies driven by different genomic aberrations. Considering the efficacy of CG'806 in the absence of observed toxicity in murine AML models, CG'806 appears superior to other FLT3 or BTK inhibitors and is suitable for development in patients with either AML or B-cell malignancy. Citation Format: Hongying Zhang, Andrea Local, Khalid Benbatoul, Peter Folger, Susan Sheng, Cheng-Yu Tsai, Stephen B. Howell, William G. Rice. CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor, targets multiple pathways to kill diverse subtypes of acute myeloid leukemia and B-cell malignancy in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 794.

Disruption of Wnt/β-Catenin Exerts Antileukemia Activity and Synergizes with FLT3 Inhibition in FLT3-Mutant Acute Myeloid Leukemia.

Purpose: Wnt/β-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases β-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/β-catenin and FLT3 inhibition in FLT3-mutant AML.Experimental Design: Wnt/β-catenin signaling was inhibited by the β-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF.Results: We found significantly higher β-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow-resident leukemic cells compared with circulating blasts. Disrupting Wnt/β-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/β-catenin and FLT3 cooperatively decreased nuclear β-catenin and the levels of c-Myc and other Wnt/β-catenin and FLT3 signaling proteins. Importantly, β-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells.Conclusions: Disrupting Wnt/β-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417-29. ©2018 AACR.

Combined inhibition of PI3Kδ and FLT3 signaling exerts synergistic antitumor activity and overcomes acquired drug resistance in FLT3-activated acute myeloid leukemia

PI3Kδ and FLT3 are frequently activated in acute myeloid leukemia (AML) and have been implicated as potential therapeutic targets. In this report, we demonstrate that combined inhibition of PI3Kδ and FLT3 exerts synergistic antitumor activity in FLT3-activated AML. Synergistic antiproliferative effects were observed in FLT3-activated MV-4-11 and EOL-1 AML cell lines, but not in FLT3-independent RS4;11 and HEL cells, as demonstrated by both pharmacological inhibition and silencing of PI3Kδ/FLT3. Combined treatment with PI3Kδ and FLT3 inhibitors more effectively inhibited AKT and ERK phosphorylation, and induced apoptosis more efficiently than either agent alone. This synergistic effect was confirmed in hematopoietic 32D cells transfected with an FLT3-ITD mutant, but not FLT3 wild type. In in vivo FLT3-activated AML xenografts, a PI3Kδ inhibitor CAL101 combined with FLT3 inhibitor led to significantly enhanced antitumor activity compared with either agent alone, in association with simultaneous inhibition of AKT and ERK. Importantly, CAL101 combined with FLT3 inhibitors overcame acquired drug resistance in FLT3-ITD AML cells. Thus, combined inhibition of PI3Kδ and FLT3 may be a promising strategy in FLT3-activated AML, particularly for patients with FLT3-inhibitor-resistant mutations.

Combined inhibition of PI3K delta and FLT3 signaling exerts synergistic antitumor activity and overcomes acquired drug resistance in FLT3-activated acute myeloid leukemia

Combined inhibition of PI3K delta and FLT3 signaling exerts synergistic antitumor activity and overcomes acquired drug resistance in FLT3-activated acute myeloid leukemia

Targeting FLT3 Signaling in Childhood Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the second most common leukemia of childhood and is associated with high rates of chemotherapy resistance and relapse. Clinical outcomes for children with AML treated with maximally intensive multi-agent chemotherapy lag far behind those of children with the more common acute lymphoblastic leukemia, demonstrating continued need for new therapeutic approaches to decrease relapse risk and improve long-term survival. Mutations in the FMS-like tyrosine kinase-3 receptor gene (FLT3) occur in approximately 25% of children and adults with AML and are associated with particularly poor prognoses. Identification and development of targeted FLT3 inhibitors represents a major precision medicine paradigm shift in the treatment of patients with AML. While further development of many first-generation FLT3 inhibitors was hampered by limited potency and significant toxicity due to effects upon other kinases, the more selective second- and third-generation FLT3 inhibitors have demonstrated excellent tolerability and remarkable efficacy in the relapsed/refractory and now de novo FLT3-mutated AML settings. While these newest and most promising inhibitors have largely been studied in the adult population, pediatric investigation of FLT3 inhibitors with chemotherapy is relatively recently ongoing or planned. Successful development of FLT3 inhibitor-based therapies will be essential to improve outcomes in children with this high-risk subtype of AML.

Skin-Specific CD301b+ Dermal Dendritic Cells Drive IL-17−Mediated Psoriasis-Like Immune Response in Mice

Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b- population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. Invivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.

Crotonoside exhibits selective post-inhibition effect in AML cells via inhibition of FLT3 and HDAC3/6.

Targeted therapies for the treatment of acute myeloid leukemia (AML), specifically the FLT3 inhibitors, have shown promising results. Nevertheless, it is very unlikely that inhibitors which target a single pathway will provide long-term disease control. Here, we report the characterization of crotonoside, a natural product extracted from Chinese medicinal herb, Croton, for the treatment of AML via inhibition of FLT3 and HDAC3/6. In vitro, crotonoside exhibited selective inhibition in AML cells. In vivo, crotonoside treatment at 70 and 35 mg/kg/d produced significant AML tumor inhibition rates of 93.5% and 73.6%, respectively. Studies on the anti-AML mechanism of crotonoside demonstrated a significant inhibition of FLT3 signaling, cell cycle arrest in G0/G1 phase, and apoptosis. In contrast to classic FLT3 inhibitor; sunitinib, crotonoside was able to selectively suppress the expression of HDAC3 and HDAC6 without altering the expression of other HDAC isoforms. Inhibitors of HDAC3 and HDAC6; RGFP966 and HPOB, respectively, also exhibited selective inhibition in AML cells. Furthermore, we established novel signaling pathways including HDAC3/NF-κB-p65 and HDAC6/c-Myc besides FLT3/c-Myc which are aberrantly regulated in the progression of AML. In addition, crotonoside alone or the combination of sunitinib/RFP966/HPOB exhibited a significant post-inhibition effect in AML cells by the inhibition of FLT3 and HDAC3/6. Inhibitors targeting the FLT3 and HDAC3/6 might provide a more effective treatment strategy for AML. Taken together, the present study suggests that crotonoside could be a promising candidate for the treatment of AML, and deserves further investigations.

Andrographolide Suppresses MV4-11 Cell Proliferation through the Inhibition of FLT3 Signaling, Fatty Acid Synthesis and Cellular Iron Uptake.

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

FLT3 Inhibitors in the Management of Acute Myeloid Leukemia.

In recent years there has been a great improvement in molecular characterization of acute myeloid leukemia (AML) allowing the stratification of patients in different rate of risk. Patients with FLT3 mutated AML have poor prognosis because of resistance to induction chemotherapy or early relapse. Several first and second generation molecules, able to inhibit FLT3 signaling have been developed and many single agent or combination studies are ongoing. Of these, quizartinib seems to have the best clinical activity. Unfortunately, resistance to FLT3 inhibitors has been observed and many scientists are currently investigating new strategy to restore sensitivity to FLT3 inhibitors.

Abstract 2364: Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling

Abstract Type III receptor tyrosine kinases (RTKs) including FLT3 and KIT play a major role in cell differentiation, proliferation, and survival of hematopoietic stem cells. FLT3-ITD and KIT-D816V mutations are the most common oncogenic mutations in FLT3 and KIT found in hematological cancers. These mutations lead to constitutive activation of proliferative and survival signals. Tyrosine kinase inhibitors (TKIs) in combination with chemotherapy display promising results in a clinical setting, but patients develop resistant disease after short-term treatment,. Hence, proteins that regulate the activity of RTKs can be alternative targets for patients carrying these mutations. Activation of FLT3 and KIT results in phosphorylation on several tyrosine residues that recruit SH2 domain-containing signaling proteins. In this study we identified Src-like adaptor protein 2 (SLAP2) as a potent FLT3 and KIT interacting protein. The interaction requires an intact SH2 domain of SLAP2 as well as phosphorylation of the receptor. Overexpression of SLAP2 in murine proB Ba/F3 cells inhibited oncogenic FLT3-ITD-mediated cell proliferation and colony formation. SLAP2 displayed a similar inhibitory potential in cells expressing KIT-D816V. SLAP2 partially blocked phosphorylation of several FLT3 and KIT downstream signaling proteins such as AKT, ERK and p38. Moreover, SLAP2 expression inhibited FLT3-ITD-mediated STAT5 phosphorylation and KIT-D816V-mediated STAT3 phosphorylation. SLAP2 expression significantly accelerated ubiquitination-mediated degradation of FLT3 and KIT. Collectively, these data suggest that SLAP2 negatively regulates FLT3 and KIT signaling and therefore, modulation of SLAP2 expression levels may become a potential target for anticancer therapy. Citation Format: Sausan A. Moharram, Lars Rönnstrand, Julhash U. Kazi. Src-like adaptor protein 2 negatively regulates FLT3 and KIT downstream signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2364. doi:10.1158/1538-7445.AM2017-2364

Abstract 1087: CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD

Abstract Introduction: CPX-351 (Vyxeos) is a liposomal combination of cytarabine and daunorubicin at a synergistic 5:1 molar ratio. Recently, CPX-351 has been shown to be significantly more effective than the 7+3 standard of care chemotherapy in treating high-risk AML patients, including patients with the FLT3-ITD mutation. We previously treated primary patient samples with CPX-351 ex vivo and found that FLT3-ITD+ samples were significantly more sensitive to CPX-351 and showed enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in an activation of liposome uptake pathways, leading to increased sensitivity to CPX-351 and ultimately cell death. Furthermore, we examined the effect of combining CPX-351 with existing FLT3 inhibitors (e.g. quizartinib and midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains an activating FLT3 mutation) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We evaluated cell viability using a colorimetric assay and measured intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry. Additionally, we analyzed the synergy of exposing these cell lines to CPX-351 and FLT3 inhibitors in combination and at different dose schedules, followed by measuring cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351 and exhibited increased drug uptake compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These data provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351. This is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Citation Format: David K. Edwards, Nathalie Javidi-Sharifi, Angela Rofelty, Max Gordon, Riley Roth-Carter, Paul Tardi, Lawrence Mayer, Jeffrey W. Tyner. CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2017-1087

The angiogenic factor Egfl7 alters thymogenesis by activating Flt3 signaling

Thymic regeneration is a crucial function that allows for the generation of mature T cells after myelosuppression like irradiation. However molecular drivers involved in this process remain undefined. Here, we report that the angiogenic factor, epidermal growth factor-like domain 7 (Egfl7), is expressed on steady state thymic endothelial cells (ECs) and further upregulated under stress like post-irradiation. Egfl7 overexpression increased intrathymic early thymic precursors (ETPs) and expanded thymic ECs. Mechanistically, we show that Egfl7 overexpression caused Flt3 upregulation in ETPs and thymic ECs, and increased Flt3 ligand plasma elevation in vivo. Selective Flt3 blockade prevented Egfl7-driven ETP expansion, and Egfl7-mediated thymic EC expansion in vivo. We propose that the angiogenic factor Egfl7 activates the Flt3/Flt3 ligand pathway and is a key molecular driver enforcing thymus progenitor generation and thereby directly linking endothelial cell biology to the production of T cell-based adaptive immunity.

Abstract 2: Aorta Intima-Resident Macrophages Contribute to Atherosclerotic Lesion Initiation

Atherosclerosis is an underlying cause of cardiovascular disease and a leading cause of mortality worldwide. Macrophage accumulation in atherosclerotic plaque, their uptake of cholesterol, and subsequent local death drive disease progression. Lipid-laden plaque macrophages are thought to be exclusively derived from blood monocyte progenitors that are recruited following endothelial damage induced by cholesterol exposure. In our current study, we focused on characterization of resident vascular macrophages that reside in the aortic intima in plaque-prone areas, previously identified as ‘vascular dendritic cells’. Using en face whole-mount confocal microscopy of aortas, we confirm a uniform resident CD64 + CD11c + CX3CR1 + MHCII + macrophage population, which is present in C57/BL6 mice resistant to atherosclerosis. Importantly, they do not express dendritic cell restricted genes zBTB46 or L-myc. We find aortic macrophages require M-CSF and Flt3 signaling for survival, but are independent of CCR2, CCR7, and GM-CSF receptor signaling, making them a distinct myeloid population. Lineage-tracing and parabiosis approaches suggest these cells derive from definitive hematopoiesis and are then self-maintained independent of blood-progenitors. Using these characterization data, we developed a labeling strategy to identify resident from recruited macrophages during kinetic studies of lesion progression. We find that resident aortic macrophages are the first cells to take up lipid following high fat diet exposure and expand within the arterial wall to form the initial lesion bed. In the absence of resident macrophages early lipid deposition in the aortic arch is ablated. Finally, utilizing an intravital carotid artery imaging approach, we identify resident aortic macrophages to be potential mediators of monocyte recruitment through direct interactions with rolling monocytes on the endothelial surface under diseased and steady-states. Overall, these results shift our understanding of the cellular mechanisms responsible for plaque construction and maintenance.

Combined inhibition of GLI and FLT3 signaling leads to effective anti-leukemic effects in human acute myeloid leukemia.

Activation of the Hedgehog pathway has been implicated in the pathogenesis of several tumor types including myeloid leukemia. Previously we demonstrated that overexpression of Hedgehog downstream mediators GLI1/2 confers an adverse prognosis to patients with acute myeloid leukemia (AML) and is correlated with a FLT3 mutated status. To analyze a possible non-canonical activation of the Hedgehog pathway via FLT3 and PI3K, we performed blocking experiments utilizing inhibitors for FLT3 (sunitinib), PI3K (PF-04691502) and GLI1/2 (GANT61) in FLT3-mutated and FLT3 wildtype AML cell lines and primary blasts. Combination of all three compounds had stronger anti-leukemic effects in FLT3-mutated compared to FLT3 wildtype AML cells in vitro. Interestingly, the colony growth of normal CD34+ cells from healthy donors was not impeded by the triple inhibitor combination possibly opening a therapeutic window for the clinical use of inhibitor combinations. Besides, combined treatment with sunitinib, PF-04691502 and GANT61 significantly prolonged the survival of mice transplanted with FLT3-mutated MV4-11 cells compared to the single agent treatments. Furthermore, the inhibition of FLT3 and PI3K resulted in reduced GLI protein expression and promotor activity in FLT3-mutated but not in FLT3 wildtype AML cell lines in western blotting and GLI1/2 promoter assays supporting our hypothesis of non-canonical GLI activation via FLT3.In summary, FLT3-mutated in contrast to FLT3 wildtype cells or normal human hematopoietic progenitor cells are exquisitely sensitive to combined inhibition by FLT3, PI3K and GLI1/2 overcoming some of the limitations of current FLT3 directed therapy in AML. The development of GLI1/2 inhibitors is highly desirable.

Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD

The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

Disruption of Wnt/β-Catenin Signaling Exerts Antileukemia Activity and Sensitizes with Tyrosine Kinase Inhibitors in FLT3 Mutated AML In Vivo

Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient.Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice.Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib).Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. DisclosuresKonopleva:Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase.Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. DisclosuresNo relevant conflicts of interest to declare.

DLX1 Affects Cell Cycle and Proliferation of Myeloid Leukemia Cells In Vitro and In Vivo

The DLX homeodomain genes are part of the Drosophila distal-less family, originally identified in the forebrain of the developing mouse embryo. DLX1 gene is expressed also in the hematopoietic cells. Our previous data showed that patients with FLT3/ITD (internal tandem duplication) mutation representing about 35% of all acute myeloid leukemia (AML) cases have higher expression of DLX1 compared to non-FLT3/ITD AML patients. Further, FLT3 signaling was described to regulate DLX1 gene expression.In the current study we found an association between DLX1 expression level and overall survival using GEP data from TCGA database within the group of FLT3/ITD-positive patients. Worse overall survival was linked to lower expression of DLX1 (p=0.003, n=46) even though generally this group of patients is characterized by their higher DLX1 level. This association was also observed when all AML patients were analyzed (p=0.01, n=197). Since the role of DLX1 in the leukemogenic process is not yet described we aimed to characterize the phenotype of leukemic cells with different expression levels of DLX1 gene.We designed two functional shRNAs (pGhU6-sh1_DLX1 and pGhU6-sh2_DLX1, referred as sh1 and sh2) to downregulate DLX1 expression in MV4;11 (FLT3/ITD positive) leukemic cells, which present high endogenous level of DLX1, and a non-silenced control (pGhU6-NSC). In vitro studies showed that cells with silenced DLX1 were arrested in G0 phase of cell cycle (35%±4.8 (NSC) to 67.5%±2.2 (sh1; p <0.01) and 65.5%±2.5 (sh2; p <0.01); flow cytometry - Pyronin Y/ Hoechst 33342 staining) and had lower proliferative activity (trypan blue over the period of 10 passages). Moreover, cells with silenced DLX1 were less apoptotic (Annexin V/PI staining).Next, we studied the impact of DLX1 downregulation on leukemic cell infiltration in vivo using sub-lethally irradiated NSG (NOD SCID gamma) mice. We injected 1x106 silenced or non-silenced MV4;11 cells via tail vein (n=20, 6 mice/group, 2 control mice). After two weeks we measured the absolute number of MV4;11 cells by flow cytometry (CD33+/GFP+/DAPI-) in bone marrow (BM) and spleen (SP) of recipient mice. Absolute cell counts of leukemic cells with silenced DLX1 were 1.26x106 (sh1; p=0.015) and 2.89x106 (sh2; p<0.0001) vs NSC cells 0.52x106 in BM and 1.59x106 (p=0.036) and 14.2x106 (p=0.025) vs 0.34x106 in SP. To ensure that the increased cell numbers were not the result of enhanced homing of DLX1 silenced cells, we performed homing experiments. Control and DLX1 silenced cells were transplanted and the number of cells in BM and SP was determined 16 hours after transplantation. We observed no differences between the studied groups, indicating that DLX1 silencing does not affect the homing ability of MV4;11 cells. Moreover, we analyzed cell cycle in leukemic cells isolated from recipient mice three weeks after transplantation. In agreement with our in vitro results, leukemic cells with silenced DLX1 had higher percentage of cells arrested in G0 phase (48.6%±6.3 (sh1; p <0.001) and 80.9%±9.3 (sh2; p <0.001) vs 26.4%±4.8 (NSC) in SP). Overall, mice with silenced DLX1 presented worse fitness and bigger splenomegaly.Further, we investigated the signaling pathways which could lead to G0 arrest. Since DLX1 inhibits TGF-β pathway through direct interaction with SMAD4, a key downstream effector of TGF-β/BMP signaling, we studied the changes in target genes in our model cell lines. Expression of CUTL-1 (sh1 - 1.5-fold, sh2 - 2-fold change to NSC), PAI-1 (3.5-fold, 4.2-fold) and CDKN1C (1.3-fold, 2.3-fold) were significantly increased in DLX1 silenced cells. PAI-1 was shown to induce replicative senescence and CDKN1C is an inhibitor of cell cycle progression. While both targets disturb cell cycle and could be responsible for the phenotype we have observed, this hypothesis needs to be elucidated in future experiments.Altogether, our data demonstrate that dysregulation of DLX1 gene in leukemic cells changes the cell phenotype. Lower level of DLX1 gene leads to arrest in G0 phase which in vitro slows down the proliferation whereas in vivo it allows the cells to persist in spleen and BM. We hypothesize that the DLX1 silenced cells become more resistant to external effects which could then be reflected as a reduced survival observed in patients with low levels of DLX1.Supported by P304/12/2214. DisclosuresNo relevant conflicts of interest to declare.