FLT3-ITD Acute Myeloid Leukemia Cell Lines Research Articles

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

HSP90 Inhibitor PU-H71 in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.

A novel lncRNA SNHG29 regulates EP300- related histone acetylation modification and inhibits FLT3-ITD AML development.

Internal tandem duplication (ITD) mutations within the FMS-like tyrosine kinase-3 (FLT3) occur in up to 25% of acute myeloid leukemia (AML) patients and indicate a very poor prognosis. The role of long noncoding RNAs (lncRNAs) in FLT3-ITD AML progression remains unexplored. We identified a novel lncRNA, SNHG29, whose expression is specifically regulated by the FLT3-STAT5 signaling pathway and is abnormally down-regulated in FLT3-ITD AML cell lines. SNHG29 functions as a tumor suppressor, significantly inhibiting FLT3-ITD AML cell proliferation and decreasing sensitivity to cytarabine in vitro and in vivo models. Mechanistically, we demonstrated that SNHG29's molecular mechanism is EP300-binding dependent and identified the EP300-interacting region of SNHG29. SNHG29 modulates genome-wide EP300 genomic binding, affecting EP300-mediated histone modification and consequently influencing the expression of varies downstream AML-associated genes. Our study uncovers a novel molecular mechanism for SNHG29 in mediating FLT3-ITD AML biological behaviors through epigenetic modification, suggesting that SNHG29 could be a potential therapeutic target for FLT3-ITD AML.

PIM Inhibition By SEL24/MEN1703 Combines Synergistically with Gilteritinib in FLT3-ITD Preclinical Models of Acute Myeloid Leukemia

BACKGROUND. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer, derived from an uncontrolled clonal proliferation of malignant myeloid blast cells in the bone marrow, transformed by recurrent genetic alterations (Wu M. et al., J. Hematol Oncol, 2018). SEL24/MEN1703 is a first-in-class, oral, type I dual PIM/FMS-like tyrosine kinase 3 (FLT3) inhibitor investigated in the ongoing Ph2 study in patients with AML (DIAMOND-01, ClinicalTrials.gov identifier: NCT03008187) (Czardybon W. et al., Oncotarget 2018). Gilteritinib is a highly potent and selective oral FLT3 inhibitor, FDA-approved for the treatment of relapsed or refractory AML with FLT3 mutations. Although Gilteritinib showed strong single-agent activity in patients with FLT3+ AML (Levis M. and Perl AE., Blood advances, 2020), the development of Gilteritinib resistance limits durability of the response, indicating that novel combination strategies may be clinically important (Zhang LS. et al., Onco Targets and Therapy, 2022). AIM. The main objective of this study is to assess potential synergistic effects in-vitro and in-vivo of the combination of SEL24/MEN1703 and Gilteritinib in AML cell lines with FLT3-ITD and to elucidate the underlying signaling pathways. METHODS.In-vitro cytotoxicity and apoptosis experiments were carried out in FLT3-ITD AML cell lines (MV4-11, MOLM13, MOLM14), testing SEL24/MEN1703 and Gilteritinib either as single agents or in combination for 72h. The combination index was calculated according to the Chou method (Chou TC., Pharmacol Rev, 2006). In-vitro co-culture experiments were performed with FLT3-ITD cell lines MV4-11 and MOLM14 incubated in the presence of human primary bone marrow stromal cells. In MV4-11 and MOLM13 xenograft models, SEL24/MEN1703 and Gilteritinib were administered per OS every day, for 14 and 22 days, respectively. A signaling pathway analysis (pAKT, pERK, pSTAT5, Mcl-1, c-Myc, Bcl-xL, pS6) was also performed through immuno-capillary electrophoresis on tumor nodules collected from MOLM13 xenograft study. RESULTS. The combination treatment was moderately synergistic/additive in both in-vitro cytotoxicity and apoptosis experiments on MV4-11, MOLM13, and MOLM14, at all concentration levels. However, in co-culture experiments, the presence of primary stromal cells resulted in a significantly increased combination index, in tested AML cell lines. In the in-vivo MV4-11 xenograft model, SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg induced a significant anti-tumor activity as single agent treatment, showing 57.2% and 84.1% of tumor volume inhibition (TVI) respectively, whereas the combination treatment SEL24/MEN1703 plus Gilteritinib induced 99.2% of TVI at day 33 (Figure 1). At day 40, when the Gilteritinib schedule was concluded, SEL24/MEN1703 and Gilteritinib as single agents showed 53.1% and 87.6% of TVI respectively, whereas the combo group induced a complete tumor regression (100% of TVI). Moreover, only the combination of SEL24/MEN1703 and Gilteritinib resulted in a delayed tumor regrowth 23.5 days after discontinuation of the treatment. In the MOLM13 model, at day 29, treatment with SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg as a monotherapy induced an antitumor activity with 38.8% and 60.1% of TVI respectively, whereas with the combination treatment resulted in a higher TVI (78.3%), however, no complete tumor regression was observed in this model. In the MOLM13 xenograft model, analysis of downstream signaling pathways of FLT3 and PIM kinases showed that the pS6 effector protein is mainly downregulated in the combination treatment group. CONCLUSIONS. Combination therapy with SEL24/MEN1703 and Gilteritinib is moderately synergistic in-vitro, however, results in robust tumor regressions, including complete responses in in-vivo FLT3-ITD models, at tolerable doses. The higher efficacy of this combination in-vivo and compared to monotherapy could be related to a very potent concomitant inhibition of FLT3 and PIM kinases affecting upregulated FLT3 signaling pathway in-vivo as demonstrated by the reduction of phosphorylation of downstream effector proteins, such as pS6. Overall, these experiments have demonstrated the antitumor potential of concomitant inhibition of FLT3 and PIM kinases which could drive a novel therapeutic strategy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Extracellular Vesicle Secretion in Combination with Venetoclax Synergistically Induces Apoptosis in FLT3-ITD+ AML

Introduction Intrinsic or acquired resistance to apoptosis is a hallmark of human cancers, including different subtypes of acute myeloid leukemia (AML). Among different mechanisms of achieving resistance, disposal of pro-apoptotic factors via extracellular vesicle (EV) release has been described for several cancer models. In AML, RNA and protein trafficking by leukemia-derived EVs has emerged as a mediator of cell-cell communication and a crucial factor in reprogramming the bone marrow (BM) niche into a leukemia permissive microenvironment. In our study, we aimed to characterize the content of primary AML-derived EVs with a special focus on apoptotic factors, and to evaluate the effect of EV release inhibition with FTY720, a FDA approved oral drug for treatment of multiple sclerosis, on apoptosis in combination with venetoclax (VEN) treatment, an inhibitor of the anti-apoptotic protein Bcl-2. Methods BM-derived EVs were purified from healthy donors (n=3), or patients with newly diagnosed AML (FLT3-WT and -ITD, n=10) after written informed consent and approval by the ethics committee in accordance with the Declaration of Helsinki. EVs were characterized by transmission electron microscopy (TEM), western blotting and nano tracking analysis (NTA). Protein and RNA from EVs were isolated and used for proteomic and RNA analyses. The apoptotic response of FLT3-WT (OCI-AML2, OCI-AML3) or FLT3-ITD (MOLM-13, MV4-11) AML cell lines and primary samples to EV release inhibitor FTY720 was assessed by flow cytometry with AnnexinV/PI. FTY720-induced EV release inhibition and intracellular EV accumulation was determined by Western Blot analysis of EV markers and visualized by immunostaining. Finally, the combination treatment of FTY720 with VEN was tested. Results TEM and immunoblotting confirmed a pure vesicle fraction (30-150 nm) with expression of characteristic markers enriched (CD63, CD81, HSP70) or absent (GAPDH, ß-actin) in EVs. NTA showed no differences in EV numbers released by AML compared to healthy cells. However, AML EVs were significantly larger in size. EV content was significantly different comparing healthy to AML-derived EVs and bioinformatic analysis from RNA sequencing revealed enrichment in pathways involved in regulation of apoptosis specifically in AML FLT3-ITD EVs (Figure 1). Of note, hsa-miR-196b-5p target interaction analysis identified BCL-2 as an exemplary anti-apoptotic target gene (Figure 2). Concordantly, AML cell lines harboring FLT3-ITD showed higher sensitivity to the EV release inhibitor FTY720 compared to FLT3-WT (IC50 = 6.2µM, 6.6µM, 13.7µM and 10.2µM respectively for MOLM-13, MV4-11, OCI-AML2 and OCI-AML3). FTY720 treatment induced intracellular EV accumulation and modulated EV marker expression confirming its effect on the EV machinery. Interestingly, FTY720 co-treatment potentiated apoptosis induced by VEN alone in both FLT3-ITD cell lines while FTL3-WT cells were still resistant to VEN and healthy hematopoiesis was unaffected. Conclusions Taken together, these results suggest that FLT3-ITD AML cells establish a protective mechanism against apoptosis by disposing of positive regulators of apoptosis via EV release. Preventing the disposal of the pro-apoptotic factors in combination with BCL-2 inhibition sensitizes AML FLT3-ITD cells to apoptosis. Additional evaluation in primary patient material and further studies to identify the factors responsible for sensitization to apoptosis upon EV release inhibition are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AD80 INHIBITS FLT3-MEDIATED SIGNALING AND HAS ANTINEOPLASTIC EFFECTS ON ACUTE MYELOID LEUKEMIA CELLS

Acute myeloid leukemia (AML) is one of the most common types of hematological malignancy in adults. Activating mutations in different tyrosine kinases have been identified as important factors in the development and progression of hematological neoplasms. For AML, FLT3-ITD has been described as a key mutation, activating signaling pathways such as PI3K, RAS, and STAT5. AD80 is a multikinase inhibitor able to inhibit the activity of kinases RET, RAF, S6K, and ERK. It is a new compound and, therefore, few studies have been carried out to date verifying its effects on tumor cells. In the present study, the cellular and molecular effects of AD80 in AML cell lines were analyzed. For the study, 6 human (FLT3-ITD: MV4-11 and MOLM13, BCR: ABL1: K562 and KU812, JAK2V617F: SET2 and HEL) and 5 murine (Ba/F3 with activating mutations: BCR::ABL1, BCR:ABL1T315I, JAK2V617F, MPLW515L, CSF3RT618I) cell lines representing different hematological neoplasms were utilized. Cell viability was assessed using MTT assay. Apoptosis, autophagy, and mitochondrial membrane potential were determined by flow cytometry. Colony formation assays were performed to analyze clonogenic ability. Protein expression/activation was assessed by western blot. In all cellular models, AD80 reduced cell viability in a time- and dose-dependent manner (all p < 0.05). MOLM13 and MV4-11 cells were the most sensitive, with IC50 ranging from 0.4 to 1.1 nM. In FLT3-ITD cell lines, AD80 induced apoptosis and autophagy, disrupted mitochondrial membrane potential, as well as decreased clonogenic ability in a dose-dependent manner (all p < 0.05). AD80 markedly decreased the activation of AKT, STAT5, S6RP, and induced PARP1 cleavage and γH2AX. AD80 was more potent in reducing the viability of FLT3-ITD AML cell lines. Indeed, it has been shown that AD80 strongly inhibits wild-type and mutated FLT3 in an in vitro assay (>90% of inhibition; Dar et al. Nature, 486:80–84, 2012). Similarly, AD80 also inhibits, in less extension, wild-type and mutated ABL1 and JAK2, which corroborate our cell viability findings. Previous studies from our group showed that AD80 has effects on AML cell lines without the tyrosine kinase-related mutations, with IC50 values ranging from 1.6 to 27.2 μM. In contrast, AD80 concentrations at 0.64 nM were able to induce apoptosis and disrupt mitochondrial membrane potential at 72 h, and induced autophagy at 24 h of treatment in FLT3-ITD AML cell lines. In the molecular scenario, this compound decreases the expression/activating of proteins related to cell growth and proliferation and increases molecular markers of autophagy and apoptosis. AD80 exhibits antineoplastic effects against FLT3-ITD mutated cell lines at nanomolar concentrations, highlighting a noticeably on-targeted effect, and opening up research possibilities that could potentially evolve to further investigations. FAPESP, CNPq, and CAPES.

P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.

The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential.

Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematopoietic stem-cell-derived leukemia with often successive derived driver mutations. Late onset acquisition of internal tandem duplication in FLT3 (FLT3-ITD) at a high variant allele frequency often contributes to full transformation to a highly proliferative, rapidly progressive disease with poor outcome. The FLT3-ITD mutation is targetable with approved FLT3 small molecule inhibitors, including midostaurin and gilteritinib. However, outside of patients receiving allogeneic transplant, most patients fail to respond or relapse, suggesting alternative approaches of therapy will be required. We employed genome-wide pooled CRISPR knockout screening as a method for large-scale identification of targets whose knockout produces a phenotypic effect that enhances the antitumor properties of FLT3 inhibitors. Among the candidate targets we identified the effect of XPO1 knockout to be synergistic with midostaurin treatment. Next, we validated the genetic finding with pharmacologic combination of the slowly reversible XPO1 inhibitor selinexor with midostaurin and gilteritinib in FLT3-ITD AML cell lines and primary patient samples. Lastly, we demonstrated improved survival with either combination therapy compared to its monotherapy components in an aggressive AML murine model, supporting further evaluation and rapid clinical translation of this combination strategy.

ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia

BackgroundIn acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with a dismal outcome. Although uncoordinated 51-like kinase 1 (ULK1), which plays a central role in the autophagy pathway, has emerged as a novel therapeutic target for various cancers, its role in FLT3-ITD AML remains elusive. In this study, we evaluated the effects of ULK1 inhibition on leukemia cell death in FLT3-ITD AML.MethodWe evaluated ULK1 expression and the levels of apoptosis and autophagy following ULK1 inhibition in FLT3-ITD AML cell lines and investigated the mechanism underlying apoptosis induced by ULK1 inhibition. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc).ResultsFLT3-ITD AML cells showed significantly higher ULK1 expression than FLT3-wild-type (WT) AML cells. Two ULK1 inhibitors, MRT 68921 and SBI-0206965, induced apoptosis in FLT3-ITD AML cells, with relatively minimal effects on FLT3-WT AML cells and normal CD34-positive cells. Apoptosis induction by ULK1 inhibition was associated with caspase pathway activation. Interestingly, ULK1 inhibition paradoxically also induced autophagy, showing synergistic interaction with autophagy inhibitors. Hence, autophagy may act as a prosurvival mechanism in FLT3-ITD AML cells. FLT3-ITD protein degradation and inhibition of the ERK, AKT, and STAT5 pathways were also observed in FLT3-ITD AML cells following treatment with ULK1 inhibitors.ConclusionULK1 is a viable drug target and ULK1 inhibition may represent a promising therapeutic strategy against FLT3-ITD AML.

Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.

The Combination of CUDC-907 and Gilteritinib Shows Promising Antileukemic Activity in Vitro and In Vivo in Preclinical Models of FLT3-ITD AML

Introduction FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations are found in approximately one quarter of acute myeloid leukemia (AML) cases. Its presence results in constitutive activation of the FLT3 receptor tyrosine kinase and its downstream growth/pro-survival pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT, and confers a poor prognosis. Gilteritinib is a selective inhibitor of FLT3 recently approved by the Food and Drug Administration for treatment of patients with relapsed/refractory AML and a FLT3 mutation. However, gilteritinib exposure induces upregulation of FLT3 - a mechanism of resistance. Previously, we showed that CUDC-907, a dual PI3K/histone deacetylase inhibitor, downregulates FLT3 expression (Li X, et al. Haematologica. 2019; epub ahead of print). We therefore hypothesized that combining CUDC-907 with gilteritinib would abrogate FLT3 upregulation and expression, resulting in synergistic antileukemic activities against FLT3-mutated AML. Methods FLT3-ITD AML cell lines and primary patient samples were treated with gilteritinib or CUDC-907, alone or in combination at clinically achievable concentrations, and subjected to annexin V/propidium iodide staining and flow cytometry analysis to quantify apoptosis. Protein levels of FLT3, Bcl-2 family proteins, and key components of the MAPK/ERK, PI3K/AKT, and JAK/STAT pathways were examined using western blotting. The impact of the observed alterations upon apoptosis were confirmed via overexpression, knockdown, and targeted inhibitor experiments. Real-time RT-PCR was used to determine FLT3 transcript levels. The FLT3-ITD AML cell line MV4-11 was used to generate a xenograft mouse model to assess in vivo efficacy of the two agents. Results CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines in vitro and patient samples ex vivo, with combined therapy. CUDC-907 abolished gilteritinib-induced expression of FLT3 in both cell lines and primary patient samples. Gilteritinib treatment reduced p-AKT, p-S6, and p-STAT5 and increased p-ERK, while CUDC-907 reduced p-AKT and p-ERK, and upregulated p-STAT5. The combination of gilteritinib and CUDC-907 decreased not only p-AKT and p-S6, but also p-ERK and p-STAT5. Targeted inhibition of ERK and JAK2/STAT5 signaling by SCH772984 and AZD1480, respectively, confirmed their roles in resistance to gilteritinib and CUDC-907 monotherapies, respectively. Combined gilteritinib and CUDC-907 treatment reduced expression of the anti-apoptotic BCL-2 family member Mcl-1 and increased expression of the pro-apoptotic protein Bim. MCL-1 overexpression and BIM knockdown partially rescued FLT3-ITD AML cells upon drug treatment, confirming their role in the antileukemic activity of combined gilteritinib and CUDC-907. To determine in vivo efficacy of the two agents, NSGS mice were injected with MV4-11 cells. Three days later, the mice were randomized into vehicle control (n=5), 40 mg/kg gilteritinib (oral gavage; n=5), 100 mg/kg CUDC-907 (oral gavage; n=5) or combination (40 mg/kg gilteritinib + 100 mg/kg CUDC-907; n=6) groups. CUDC-907 was given daily for 5 days on, 2 days off, for a total of 4 cycles. Gilteritinib was administered daily for 28 days. Both agents were well tolerated; maximal weight loss was 5.5%, 0.9%, and 6.7% in the CUDC-907, gilteritinib, and combination groups, respectively. Median survival of mice in the vehicle control group was 43 days. Median survival in the CUDC-907 monotherapy and gilteritinib monotherapy arm was 40.5 days and 104 days, respectively. One mouse in the combination therapy arm died on day 138, while the remaining 5 mice in the combination therapy arm continue to survive, as of time of writing (day 168), and are asymptomatic (Figure 1). Conclusion We confirmed that the combination of CUDC-907 plus gilteritinib synergistically induces apoptosis in both FLT3-ITD AML cell lines and primary patient samples, and that gilteritinib-induced FLT3 expression is abolished by CUDC-907. Cooperative inhibition of the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, as well as upregulation of Bim/downregulation of Mcl-1 all appear to contribute to this observed antileukemic synergy. Our cell line-derived xenograft mouse model provides strong evidence of in vivo efficacy and robust grounds for clinical translation of this therapeutic combination. Disclosures No relevant conflicts of interest to declare.

LAM-003, a Novel Oral Heat Shock Protein 90 Inhibitor for Treatment of Acute Myeloid Leukemia, Including Wild-Type and FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Disease

Acute myeloid leukemia (AML) remains a disease with high unmet medical need. While most patients respond to initial therapy, few are cured, relapse rates are high, and most patients eventually develop life-threatening complications. FLT3-mutant disease is a particularly aggressive subtype. Recent approval of drugs targeting FLT3-mutant disease have improved short-term outcomes but not all patients respond, and duration of response is limited by secondary mutations that impede FLT3 inhibitor (FLT3i) binding (intrinsic factors) and by secreted stromal factors that activate alternative pro-survival pathways (extrinsic factors). Heat-shock protein 90 (HSP90) is a chaperone protein involved in many cellular processes and inhibition of HSP90 can have pleiotropic effects in targeting cancer cells such as degradation of oncoproteins that drive survival and proteins that mediate protective signaling. Here, we describe the nonclinical activity of LAM-003, an orally bioavailable HSP90 inhibitor (HSP90i) under clinical development for AML. To assess the anti-leukemic activity of LAM-003, we tested a panel of AML cell lines and primary AML samples. LAM-003 inhibited proliferation of both FLT3-mutant and wild-type cell lines, with preferential activity against cells harboring FLT3-ITD (geometric mean FLT3-ITD EC50 = 670 nM [n=8] vs FLT3 WT EC50 = 1400 nM [n=16]). Additionally, we observed that LAM-003 was potent in a subset of the FLT3 WT cells. To explore whether LAM-003 was effective against tumors driven by oncoproteins that are client proteins of HSP90, we focused on AML cells harboring FLT3-ITD. We confirmed that LAM-003 reduced cell surface FLT3-ITD expression and downstream signaling in MV-4-11 and MOLM-13 cells, consistent with HSP90i-mediated degradation of FLT3-ITD. In BA/F3 cells expressing FLT3-ITD with various secondary resistance mutations, we observed that LAM-003 elicited a dose-dependent reduction of FLT3 mutant cell surface expression. Moreover, BA/F3 cells expressing FLT3-ITD and the F691L mutation exhibited the expected resistance to crenolanib, yet LAM-003 retained anti-proliferative activity. Additionally, MOLM-13 cells harboring a FLT3 D835Y mutation demonstrated expected resistance to the FLT3i sorafenib and tandutinib yet remained sensitive to LAM-003. Finally, primary AML blasts harboring a D835 mutation displayed sensitivity to LAM-003 when tested ex vivo. To evaluate the potential of LAM-003 to overcome bone-marrow-stroma-derived resistance, FLT3-ITD AML cell lines (MV-4-11, MOLM-13, MOLM-14) were assayed in unconditioned or stromal-cell-conditioned medium. Conditioned medium dramatically reduced the potency of FLT3i but LAM-003 demonstrated equal potency under both conditions. We also showed that stromal cell co-culture induced FLT3i resistance in MOLM-13 cells whereas LAM-003 retained potent activity. Recognizing that the inherent genetic heterogeneity of AML blasts limits the curative potential of a single drug, we performed in vitro studies to identify drugs that synergize with LAM-003 in 3 FLT3-ITD AML cell lines. Synergy was demonstrated with FLT3i, daunorubicin, azacitidine or cytarabine, with the most robust synergy being observed with venetoclax. Extending the evaluation to AML cells wild type for FLT3 and cell lines from other hematologic indications (multiple myeloma, diffuse large B-cell lymphoma and mantle cell lymphoma), we found that the synergy was not limited to cells harboring FLT3-ITD, but rather correlated with BCL-2 abundance, suggesting a fundamental mechanism of action that depends on BCL-2 family-mediated survival. Mechanistic studies demonstrated that the combination of LAM-003 and venetoclax inhibited AKT-mediated regulation of GSK3B, resulting in MCL-1 degradation. In vivo studies using a MOLM-13 systemic model of FLT3-ITD AML demonstrated that LAM-003 monotherapy significantly improved animal survival and that the combination of LAM-003 and venetoclax significantly prolonged animal survival compared with each single agent. These nonclinical studies demonstrate that LAM-003 exhibits antileukemic activity, overcomes mechanisms of FLT3i resistance and potently synergizes with existing AML drugs. As such, our data provide strong rationale for evaluation of LAM-003 in an ongoing clinical trial in patients with AML (NCT03426605). Disclosures Beeharry: AI Therapeutics: Employment, Equity Ownership. Landrette:AI Therapeutics: Employment. Grotzke:AI Therapeutics: Employment. Gayle:AI Therapeutics: Equity Ownership. Young:AI Therapeutics: Employment, Equity Ownership. Miller:Incuron, Inc.: Consultancy; Cleveland Biolabs, Inc: Employment, Equity Ownership; Calistoga Pharmaceuticals, Inc.: Equity Ownership; AI Therapeutics: Consultancy, Equity Ownership; VelosBio Inc.: Employment, Equity Ownership; Acerta Pharma, Inc.: Equity Ownership. Xu:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rothberg:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lichenstein:AI Therapeutics: Employment, Equity Ownership.

AXL/Mertk Inhibitor ONO-7475 Potently Synergizes with BCL2 Inhibitor ABT-199, Overcomes ABT-199 Resistance Mechanisms, and Kills FLT3 ITD AML Cells

AXL/Mertk Inhibitor ONO-7475 Potently Synergizes with BCL2 Inhibitor ABT-199, Overcomes ABT-199 Resistance Mechanisms, and Kills FLT3 ITD AML Cells

Novel Therapy for FLT3-ITD Acute Myeloid Leukemia Utilizing the Combination of CUDC-907 and Gilteritinib

Introduction:FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase, and is mutated in approximately one third of acute myeloid leukemia (AML) patients; this mutation confers a poor prognosis. Two FLT3 mutations are commonly seen in AML: internal tandem duplications (ITD) in the juxtamembrane domain (~25% of AML), and point mutations in the receptor tyrosine kinase at codon 835 (D835) (~7% of AML). Both mutations result in constitutive FLT3 activation, causing downstream activation of multiple pathways, in particular, those involved in cell survival including the RAS-RAF-MEK-ERK, JAK-STAT5, and PI3K/AKT pathways. PI3K-AKT may also be activated by AXL, also a tyrosine kinase, via its targets PLC, Grb2, and PI3K. Logically, then, inhibition of FLT3 is a promising pharmacological approach for treating this subtype of AML. Gilteritinib (ASP-2215) is a novel dual inhibitor of FLT3 and AXL, exposure to which results in upregulation of FLT3 as a resistance mechanism. Previously, we found that the novel dual PI3K/histone deacetylase (HDAC) inhibitor CUDC-907 downregulates FLT3 expression in AML cells (Figure 1A). Additionally, inhibition of FLT3 and AXL by gilteritinib may not result in robust inactivation of both the PI3K-Akt and MEK/ERK pathways due to crosstalk between the two pathways. Thus, our hypothesis was that CUDC-907 would sensitize AML cells to gilteritinib, resulting in concurrent inhibition of all the downstream signaling pathways of FLT3 and AXL, leading to synergistic antileukemic activities again FLT3-mutated AML (Figure 1B).Methods:FLT3-ITD AML cell lines (MV4-11 and MOLM-13) and primary patient samples were treated with CUDC-907, gilteritinib, both, or neither for 24 hours, at clinically achievable concentrations. Annexin V/Propidium Iodide (PI) staining and flow cytometry analyses was performed, and combination indexes (CI) calculated; CI<1, CI=1, and CI>1 indicating synergistic, additive, or antagonistic effects, respectively. Western blots were performed after treatment for 0-24 hours to determine protein expression of relevant targets.Results:CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines and FLT3-ITD patient samples (AML#171, AML#180), the combination exceeding either in isolation (Figure 1C). These findings were confirmed via western blot, which showed accentuated upregulation of cleaved caspase3 with combination therapy, in both cell lines and one patient sample, demonstrating drug-induced apoptosis. We confirmed that CUDC-907 abolishes gilteritinib-induced expression of FLT3 in a time-dependent fashion in cell lines MV4-11 and MOLM-13 (Figure 1D). Gilteritinib treatment decreased p-AKT, p-S6, and p-STAT5, while inhibition of the ERK pathway, as assessed by p-ERK expression, varied amongst the samples (Figure 1E). CUDC-907 treatment decreased both p-AKT and p-ERK. MOLM-13 cells showed increased p-ERK following gilteritinib treatment and increased p-STAT5 after CUDC-907 treatment. In all samples, combination of gilteritinib with CUDC-907 resulted in decrease of p-STAT5 and p-S6, similar to gilteritinib treatment alone, and further reduction of p-AKT and p-ERK compared to single drug treatments. Gilteritinib treatment also reduced expression of anti-apoptotic protein Mcl-1, which was further decreased in combination treated cells. Subsequently, time-course analysis was performed in both cell lines; findings were consistent with prior observations, and confirmed that protein expression changed over time, in relation to gilteritinib/CUDC-907/combined treatment exposure.Conclusion:We confirmed that CUDC-907 and Gilteritinib synergistically induce apoptosis in both cell lines and primary patient samples derived from patients with FLT3-ITD AML, and that CUDC-907 abolishes Gilteritinib-induced FLT3 expression. Additionally, the combination cooperatively inhibits the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, while preventing escape via alternative pathways. Our results provide a strong foundation for subsequent in vivo murine studies, and eventual clinical evaluation of the combination of gilteritinib and CUDC-907 for the treatment of AML. [Display omitted] DisclosuresGe:MEI Pharma: Research Funding.

Antileukemic Activity of 8-Chloro-Adenosine (8-Cl-Ado) Is Mediated By Mir-155 Degradation and ErbB3 Binding Protein (Ebp1)-Dependent p53 Activation: A Novel Therapeutic Approach for FLT3-ITD Acute Myeloid Leukemia (AML)

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016).Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. [Display omitted] DisclosuresWennerberg:Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

Concurrent Inhibition of Pim-1 and FLT3 Kinases in FLT3-ITD Acute Myeloid Leukemia Post-Translationally Downregulates the Anti-Apoptotic Protein Mcl-1 through Downregulation of the Mcl-1 Deubiquitinase USP9X

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors.The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells.Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14.Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control.The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation.Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. DisclosuresTron:AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.

Abstract 5408: The novel retinamide VNLG-152, which targets translation by promoting degradation of MAPK-interacting kinases, has preferential activity in acute myeloid leukemia with FLT3-ITD

Abstract Purpose: Acute myeloid leukemia (AML) with internal tandem duplication (ITD) mutations of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase has a poor prognosis, with short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. FLT3 inhibitors are active, but their activity is limited and transient. New treatments and approaches are needed. The novel retinamide (NR) VNLG-152, targets translation by promoting degradation of MAPK-interacting kinases (Mnks), thereby inhibiting phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), a downstream target of FLT3-ITD. eIF4E aids in translation by recruiting ribosomes to mRNA, a process that is upregulated in AML with FLT3-ITD. The association of the eIF4E protein complex is the rate- limiting step in translation initiation. We characterized the effects of VNLG-152 in AML cell lines and patient samples with FLT3-ITD and with wild-type FLT3. Methods: Cell lines studied included human MV4-11 and MOLM-14, with endogenous FLT3-ITD expression, HL60 and U937, with wild-type FLT3, and Ba/F3-ITD and Ba/F3-WT, which are murine Ba/F3 cells transfected with human FLT3-ITD and wild-type FLT3, respectively. Cytotoxicity was measured in a WST-1 colorimetric assay, proliferation by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, and apoptosis by Annexin V-FITC and propidium iodide labeling, measured by flow cytometry. AML patient samples obtained on an IRB-approved protocol were studied in cytotoxicity assays. Results: VNLG-152 was cytotoxic to the human FLT3-ITD AML cell lines MV4-11 and MOLM-14 with low micromolar IC50 concentrations, while IC50 concentrations were greater than 10 μM in the wild-type FLT3 AML cell lines HL60 and U937. IC50s in Ba/F3-ITD and Ba/F3-WT cells were 3.6 μM (95% CI, 3.0-3.9 μM) and 5.7 μM (95% CI, 5.0-6.4 μM), respectively. The cellular effects of VNLG-152 were evaluated further in Ba/F3-ITD cells. Concentration-dependent slowing of proliferation was seen at low micromolar concentrations, while concentration-dependent pro-apoptotic effects occurred at higher concentrations. VNLG-152 was also cytotoxic to blasts isolated from FLT3-ITD AML patient samples at IC50 concentrations &amp;lt;1 μM, whereas its IC50 concentrations in blasts from AML patient samples with wild-type FLT3 were &amp;gt; 5 μM. Conclusion: We conclude that the novel Mnk degrading agent VNLG-152 is active in AML with FLT3-ITD. Subsequent work will examine combinations with FLT3 inhibitors and with chemotherapy drugs in this prognostically unfavorable AML subtype. Citation Format: Sheetal Karne, Karthika Natarajan, Senthilmurugan Ramalingam, Lalji K. Gediya, Vincent C.O. Njar, Maria R. Baer. The novel retinamide VNLG-152, which targets translation by promoting degradation of MAPK-interacting kinases, has preferential activity in acute myeloid leukemia with FLT3-ITD. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2015-5408

The FLT3 and PDGFR inhibitor crenolanib is a substrate of the multidrug resistance protein ABCB1 but does not inhibit transport function at pharmacologically relevant concentrations.

Background Crenolanib (crenolanib besylate, 4-piperidinamine, 1-[2-[5-[(3-methyl-3-oxetanyl)methoxy]-1H-benzimidazol-1-yl]-8-quinolinyl]-, monobenzenesulfonate) is a potent and specific type I inhibitor of fms-like tyrosine kinase 3 (FLT3) that targets the active kinase conformation and is effective against FLT3 with internal tandem duplication (ITD) with point mutations induced by, and conferring resistance to, type II FLT3 inhibitors in acute myeloid leukemia (AML) cells. Crenolanib is also an inhibitor of platelet-derived growth factor receptor alpha and beta and is in clinical trials in both gastrointestinal stromal tumors and gliomas. Methods We tested crenolanib interactions with the multidrug resistance-associated ATP-binding cassette proteins ABCB1 (P-glycoprotein), ABCG2 (breast cancer resistance protein) and ABCC1 (multidrug resistance-associated protein 1), which are expressed on AML cells and other cancer cells and are important components of the blood-brain barrier. Results We found that crenolanib is a substrate of ABCB1, as evidenced by approximate five-fold resistance of ABCB1-overexpressing cells to crenolanib, reversal of this resistance by the ABCB1-specific inhibitor PSC-833 and stimulation of ABCB1 ATPase activity by crenolanib. In contrast, crenolanib was not a substrate of ABCG2 or ABCC1. Additionally, it did not inhibit substrate transport by ABCB1, ABCG2 or ABCC1, at pharmacologically relevant concentrations. Finally, incubation of the FLT3-ITD AML cell lines MV4-11 and MOLM-14 with crenolanib at a pharmacologically relevant concentration of 500nM did not induce upregulation of ABCB1 cell surface expression. Conclusions Thus ABCB1 expression confers resistance to crenolanib and likely limits crenolanib penetration of the central nervous system, but crenolanib at therapeutic concentrations should not alter cellular exposure to ABC protein substrate chemotherapy drugs.

Synergistic Antileukemic Effect Of Sequential Administration Of Dichloroacetate (DCA) Combined With Arsenic Trioxide (ATO) In Primary Blasts From Patients With Acute Myeloid Leukemia (AML) and FLT3-ITD AML Cell Lines

BackgroundOutcomes of treatment for AML are suboptimal with ara-C and anthracyclines particularly in AML with internal tandem duplication (ITD) mutation of the fms-like tyrosine kinase 3 (FLT3-ITD), which is known for hyperproliferative state with activation of glycolytic metabolism and increased intracellular reactive oxygen species (ROS). ATO is highly effective in treatment of acute promyelocytic leukemia (APL). In addition to degradation of PML-RARα in APL, ATO has multiple targets within the cell including reduction of phosphorylation of STAT proteins (downstream of FLT3) and decreasing mitochondrial membrane potential leading to apoptosis. By inhibiting pyruvate dehydrogenase kinase, DCA is known to shunt pyruvate away from lactate production and into mitochondrial respiration, resulting in enhanced oxidative phosphorylation. DCA has been used clinically for treatment of lactic acidosis in children and adults. We hypothesized that treating leukemic cells, particularly with FLT3-ITD mutations, for short periods of time with sublethal dose of DCA (priming) might thus activate mitochondria to produce more ROS on exposure to ATO, resulting in enhanced anti-leukemic activity. MethodsIC50s for ATO and DCA were generated in AML cell lines with FLT3-ITD (MOLM14, and MV4-11), with FLT3 point mutation (MonoMac6), and with FLT3-WT (THP-1), and normal karyotype FLT3-WT primary AML cells (AML13, AML15, AML16). To generate potentiation factors, cells were primed with IC30 of DCA or vehicle for 48 h and then treated with a range of ATO concentrations in the presence or absence of DCA (IC30) for an additional 48 h. Primary cells were treated with DCA or vehicle for 24 h and then the combination for 24 h. Each experiment was terminated with WST1 (Roche). Cell viability assays were carried out similarly except the endpoint was trypan blue exclusion. Combination Index (CI) was calculated by the fixed ratio method of Chou and Talalay. Apoptosis was monitored using the FITC Annexin V Kit (BD Pharmingen) while mitochondrial potential was assessed by MitoRed Kit (Millipore); cells were analyzed by FACScan (BD Biosciences) and Flow Jo Software (Tree Flo). Numbers are mean ± SD (triplicates). ResultsIC50s of ATO and DCA alone, and when ATO is combined sequentially with IC30 of DCA are shown in Table 1. Presence of DCA increased the cytotoxicity of ATO by 1.3- to 2.2-fold (potentiation factor), and decreased cell viability by approximately 45-95% only in cell lines with FLT3 mutations (MOLM-14, MV4-11, MonoMac6). Cytotoxicity of ATO in FLT3-WT cells, THP-1, was not potentiated with DCA because cells were highly sensitive to DCA alone. A clear synergistic, but not additive, anti-leukemic effect (CI< 1.0) was observed with DCA and ATO in FLT3 mutated cell lines. When MOLM-14 cells were primed with DCA for 24 h and treated with ATO at IC50 for 48 h, an 83% increase in apoptosis (p=0.002), and a 55% increase in loss of mitochondrial membrane potential (p=0.003) were observed compared to no priming. With no DCA priming and combination of ATO+DCA for 48 h, apoptosis and loss of mitochondrial membrane potential were increased by 50% (p= 0.003), and 120% (p=0.008), respectively, compared to ATO alone. To a lower extent, the same effect was observed in primary AML cells. ConclusionIn AML cells with FLT3 mutations, priming with sublethal dose of DCA, as a mitochondrial enhancer, significantly potentiated the cytotoxicity of ATO. Mechanistic studies on FLT3 signaling in primary AML cells with FLT3-ITD are ongoing. These data suggest that targeting cellular metabolism in leukemia may provide a chemotherapy-free therapeutic option for AML patients who are medically unfit or patients with unfavorable risk such as relapsed/refractory FLT3-ITD and warrants further clinical investigation. Disclosures:Off Label Use: Arsenic trioxide in AML.