Cell Lines MOLM-13 Research Articles

Notch Activation Rescues Exhaustion in CISH-Deleted Human iPSC-Derived Natural Killer Cells to Promote In Vivo Persistence and Enhance Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS) is a critical negative regulator of IL-15 signaling in natural killer (NK) cells and is encoded by CISH gene in human. Recent studies in a murine model demonstrate that CIS is a potent inhibitory checkpoint in NK cell-mediated tumor immunity. However, it is unclear if CIS similarly regulates human NK cell-mediated anti-tumor activity. Unlike CTLA-4 and PD1 that are expressed at the cell surface and can be blocked by antibody-mediated therapy, CIS is expressed intracellularly. Therefore, we deleted CISH gene in human induced pluripotent stem cells (iPSCS) using CRISPR/Cas9 technology and characterized the CISH-knockout iPSC lines. Our study demonstrates that CISH does not regulate maintenance of undifferentiated human pluripotent stem cells. We then derived NK cells from these gene-modified iPSCs using a two-stage in vitro hematopoietic-lymphoid differentiation protocol developed by our group. Initial hematopoietic differentiation of the iPSCs was unaffected by CISH deletion. However, deletion of CISH in iPSCs markedly delayed the second stage of in vitro NK cell differentiation. Specifically, whereas NK cell differentiation was typically fully complete with >90% NK cells after 4 weeks using WT iPSCs, the CISH-/- iPSC- cells only produced ~10% CD45+CD56+ NK cells at 4 weeks, though by 5 weeks these cultures were >80% NK cells. The CISH-/- iPSC-derived NK cells demonstrated typical NK surface maker expression, including CD94, CD16, NKG2D, NKp44, NKp46, FasL, and KIRs. Initial studies demonstrated that CISH-/- human iPSC-derived NK cells had significantly reduced ability to expand in vitro with evidence of NK cell exhaustion, including increased TIM-3 expression, decreased IFN-γ production and decreased cytotoxicity. RNA-seq analysis also confirmed that expression of exhaustion-related genes, including TIM3, CTLA4, and 2B4, were all significantly increased in CISH-/- NK cells. Interestingly, mass cytometric (CyTOF) analysis of CISH-/- NK cells with a panel of 36 phenotypic and functional NK cell markers identified an exhausted sub-population (increased expression of exhaustion marker TIM3 and inhibitory receptors such as ILT2 and Siglec7, as well as decreased proliferation marker Ki67). Importantly, this exhaustion could be rescued by co-culturing CISH-/- NK cells with Notch ligand-expressing OP9-DL4 stromal cells, leading to production of fully functionally mature NK cells. The CISH-/- NK cells stimulated by Notch maintained better expansion and improved cytotoxic function with low concentrations of either IL15 or IL2 compared to WT-iPSC-derived NK cells. CISH-/- NK cells demonstrate increased cytotoxic activity against leukemia cell lines K562 and MOLM-13 cells in vitro. Moreover, single-cell cytokine response analysis of CISH-/- NK cells after Notch stimulation showed >10 fold enrichment of polyfunctional cell subsets with effector cytokine production (Granzyme B, IFN-γ, MIP-1α, Perforin, TNF-α) compared with WT NK cells or NK cells from peripheral blood. In a MOLM-13 xenograft model, CISH-/- NK cells displayed significantly increased persistence in peripheral blood in comparison with WT NK cells (CISH-/- NK cells 6.81 ± 0.85 % vs WT NK cells 2.05 ± 0.25 N=5). More importantly, CISH-/- NK cells show significantly better control of tumor progression in the MOLM-13 xenograft model compared with WT NK cells. Together, these studies demonstrate CISH plays a key role to regulate NK cell activation-induced exhaustion and that Notch activation prevents this exhaustion to enable production of functionally hyperactive NK cells. DisclosuresGuan:Vivace: Equity Ownership. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

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.

Combinatorial Approaches to Overcome Plasma Protein Inhibition of FLT3 Tyrosine Kinase Inhibitors

Background: The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML) and also results in poor prognosis for adult and pediatric patients, and thus represents an attractive target for tyrosine kinase inhibitors (TKI). The activity of FLT3-targeted TKI is inhibited to varying extents by plasma protein binding. Staurosporine (STS)-derived TKI are inhibited almost exclusively by the plasma protein alpha-1 acid glycoprotein (AGP), an acute-phase reactant. We studied the impact of AGP binding on the other STS-derivatives and report the development of a novel method to overcome this binding.Methods: We assayed the impact of human AGP upon the activity of the STS-derived TKI (midostaurin, lestaurtinib, TTT-3002) against proliferation of the FLT3-ITD dependent cell line MOLM-14 and upon the parent compound (staurosporine) against the non-FLT3-dependent cell line HL-60. These experiments were repeated, co-incubating with drugs that competitively bind AGP to identify those that may restore TKI activity.Results: The TKI are inhibited in a linear AGP-dependent manner (fold change increase of IC50 per mg/dL AGP: midostaurin 3.00-fold, lestaurtinib 11.73-fold, TTT-3002 0.33-fold) across the range of AGP concentrations observed in human plasma. These results correspond to the drug-protein binding constants for the TKI: midostaurin 12.6 µM-1, lestaurtinib 49.2 µM-1, TTT-3002 1.41 µM-1, all validated by competitive fluorescence displacement of the AGP-binding dye, 1-anilino-8-naphthalenesulfonate. These results predict that in vivo IC50 values for these FLT3 TKI will be significantly higher than those measured under typical (10% FCS) in vitro culture conditions: midostaurin 4.7 µM, lestaurtinib 4.8 µM, TTT-3002 34 nM. By comparison, activity of the parent compound, staurosporine, against HL60 is completely inhibited by AGP. Assays using bovine plasma, serum or purified AGP do not demonstrate similar inhibition of FLT3 TKI. We are developing a murine model to overcome this experimental limitation.We have developed a mathematical model describing the interactions of AGP with FLT3 TKI using classical mass action relationships that match experimental results and furthermore describe the effects of competitive plasma protein binding by unrelated agents. These models predict that disinhibition of TKI may be achievable in vivo, and define the properties of such “rescue” agents.Mifepristone binds AGP (2-10 fold greater than STS-derived TKI) and has no independent effect upon FLT3-dependent cell growth. Co-treatment with mifepristone restores the IC50 of TTT-3002 from 12 nM with AGP to < 0.1 nM. Disinhibition is seen for lestaurtinib (IC50 shift reduced from >1000-fold to 50-fold) and midostaurin (300-fold reduced to 80-fold). This results in predicted in vivo IC50 that are clinically relevant, and serve as a proof-of-principle for this method. Using this principle we have screened a library of FDA-approved compounds for the ability to rescue TKI activity despite the presence of potentially inhibitory plasma proteins. This screen has identified 40 potential agents that may displace STS-derived TKI from AGP, and an additional 90 agents that may restore TKI activity through off-target effects. Several agents have already been validated in vitro, and found to decrease the IC50 of midostaurin and other TKI to clinically achievable ranges despite the presence of inhibiting proteins.Conclusions: The failure of FLT3 TKI in previous clinical trials has been linked to a lack of plasma drug activity. This work provides biochemical confirmation of this effect. Furthermore, these results indicate that this is a property of the class as a whole, including midostaurin. Indeed, for midostaurin, the predicted in vivo IC50 is higher than steady state levels suggesting that in clinical trials it likely acts through non-FLT3 mechanisms. Disinhibition of TKIs by mifepristone suggests a novel combinatorial approach restore TKI activity using unrelated compounds. We are currently examining other agents for similar synergy. By improving TKI potency in the face of inhibitory plasma protein binding, such combinations would be expected to improve their clinical efficacy by reducing the dosages necessary to thoroughly inhibit FLT3. Finally, this report provides a method for predicting at least one factor that affects the success or failure of FLT3 TKI in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

NGS-Based Monitoring of Measurable Residual Disease in FLT3-ITD Positive Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), most patients respond to chemotherapy initially, but the risk of relapse remains high. Continued monitoring of measurable residual disease (MRD) following therapy onset enables early assessment of treatment response and clinical intervention. However, while appropriate assays exist for several genetic markers, some of the most common aberrations in AML, FLT3 internal tandem duplications (FLT3-ITDs), have remained a challenging target due to insertion site and length heterogeneity. The established diagnostic assay based on PCR and fragment analysis (FA), on the other hand, lacks sufficient sensitivity. In consequence, extensive monitoring of FLT3-ITD MRD in AML patients over time and treatment is not yet available. At the same time, with the approval of the FLT3-targeting tyrosine kinase inhibitor (TKI) midostaurin, the need for detailed monitoring of FLT3-ITD clones has increased.Aims: We have developed a new next-generation sequencing (NGS) based assay and analysis program to detect FLT3-ITDs with high sensitivity and specificity. We aimed to demonstrate our method's applicability to MRD monitoring and evaluated the progression of FLT3-ITD MRD in patients treated with midostaurin within the AMLSG 16-10 trial.Methods: FLT3 exons 14 and 15 were PCR-amplified and sequenced on the Illumina MiSeq to generate at least 1 million 250 bp paired-end reads. ITDs were detected using our novel analysis program that performs all steps of the analysis, does not require any manual filtering and reports ITDs extensively annotated to the user. To assess our assay's concordance with established methods, FLT3-ITDs in all diagnosis and relapse samples were characterized by FA and Sanger sequencing (SSeq).Results: Assay specificity was confirmed with 0 ITDs reported in four FLT3-ITD⁻ control samples (healthy volunteers n=2, AML patient n=1, AML cell line HL60 n=1). To assess assay sensitivity, we serially diluted DNA of the ITD⁺ AML cell line MOLM14 in DNA of the ITD⁻ AML cell line HL60. The expected 21 bp ITD was detected in all four dilutions, down to a variant allele frequency (VAF) of 6.7x10⁻⁵ (0.0067 %). We further processed matched diagnosis and relapse samples from 9 ITD⁺ AML patients at low coverage (mean: 430000 paired-end reads) and confirmed concordance of ITD site, length (range 21-198 bp) and VAF with results from FA/SSeq. For 4/9 patients, we independently sequenced a total of 12 matched diagnosis, remission and relapse samples to our target coverage of 1.5 million paired-end reads. In all of these, ITDs detected by FA were confirmed and ITD site, length and VAF were again concordant between FA/SSeq and our NGS-based approach; results from the diagnosis and relapse samples sequenced to both low and high coverage were highly reproducible.In 11/12 samples sequenced to high coverage, our assay identified additional ITD clones that were not identified by FA: The mean number of ITDs detected was 4.8 at diagnosis (1.5 by FA), 2.0 at clinical remission (not measured by FA) and 1.5 at relapse (0.5 by FA). VAFs of these ITDs ranged from 0.007-38 % (mean 5.3 %); ITDs that were solely detected by our NGS-assay were present at lower VAFs than those also identified by FA/SSeq (NGS-assay only: mean 0.21 %, range 0.007-2 %; also detected by FA/SSeq: mean 21.4 %, range 2-38 %). About half of the ITDs detected by NGS at diagnosis (11/19 ITD clones) were not detected at any of the later time points and thus presumably eradicated by treatment. Conversely, 8/19 ITD clones persisted at remission (6/19) or reoccurred at relapse (2/19). In fact, nearly all of the ITD clones present at relapse were already identified at an earlier time point.Conclusion: In sum, we could demonstrate that our NGS-based assay and analysis program achieve high accuracy and precision and enable MRD monitoring in FLT3-ITD positive AML patients. Its high sensitivity enables the detection of additional ITD clones occurring at low allelic frequencies not only at remission but also at diagnosis and relapse. Further evaluation of MRD monitoring in FLT3-ITD positive AML patients treated with midostaurin within the AMLSG 16-10 trial is currently ongoing. DisclosuresBullinger:Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Research Funding, Speakers Bureau.

TAM Inhibitor ONO-7475 Combination with Sorafenib Is Effective Using in Vitro and In Vivo Models of FLT3 ITD Acute Myeloid Leukemia

Background: Internal tandem duplication (ITD) mutation in Fems-like kinase 3 (FLT3) occur in ~ 25% of newly diagnosed acute myeloid leukemia (AML) patients and are associated with poor survival outcome. We recently demonstrated that highly specific AXL/MER inhibitor ONO-7475 (Ono Pharmaceutical Co, Osaka, Japan) was effective as a single agent against FLT3 ITD AML cells studied with in vitro co-culture and murine xenograft models (Ruvolo et al Haematologica, 2017). Based on expression of TAM kinases in the AML cells tested, mechanism for ONO-7475 killing in the FLT3 ITD AML cells was due to AXL inhibition. Here we report on the in vitro and in vivo effects of ONO-7475 when combined with FLT3 inhibitor Sorafenib in FLT3 ITD AML models.Methods: FLT3 ITD AML cell lines Molm13 and MV4;11 as well as Sorafenib resistant Molm13 cells were treated with varying doses of ONO-7475 in the presence and absence of FLT3 inhibitor Sorafenib and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. RNA was isolated and gene expression profiling (GEP) analysis using microarray was performed on ONO-7475 treated cells. GEP results for a number of genes including CDK1, PLK1, and other cell cycle regulators were validated by qRT-PCR. The efficacy of ONO-7475 combination with Sorafenib in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Both drugs were given by oral gavage (ONO-7475 at 10 mg/kg and Sorafenib at 5 mg/kg). Drugs were given 5 days a week. Leukemia burden was assessed by IVIS imaging.Results: Molm13 and MV4;11 cells were highly sensitive to ONO-7475 combination with Sorafenib. A dose of 50 nM ONO-7475 with 25 nM Sorafenib potently induces apoptosis and eliminates > 90% of cells after 72 hours. Sorafenib resistant Molm13 cells were more resistant to either drug compared to parental cells. However, combination of 50 nM ONO-7475 with 100 nM Sorafenib potently induces apoptosis in the Sorafenib resistant cells with nearly all leukemic cells eliminated after 72 hour treatment. GEP analysis of cells treated with the AXL inhibitor revealed suppression of many genes involved in cell cycle control including various CDKs (e.g. CDK1, CDK4), Cyclins (e.g Cyclin B1), and other cell cycle regulators such as PLK1. ONO-7475 inhibition of gene expression of these genes was verified by qRT-PCR. GEP also revealed induction of genes associated with more mature myeloid cells including HLA-DR, CD114, and CD115. Finally, single agent use of ONO-7475 (10 mg/kg) or Sorafenib (5 mg/kg) had limited effect in the FLT3 ITD AML xenograft model; however, the combination of both drugs at single agent dose was effective reducing leukemia burden and significantly enhancing survival of mice bearing the Molm13 leukemia cells.Conclusions: These results suggest that ONO-7475 combination with Sorafenib is effective killing AML cells with FLT3 ITD including those that are resistant to the FLT3 inhibitor. The identification of gene expression of cell cycle regulators as novel targets of the drug suggests for the first time that AXL regulates cell cycle via a complex transcriptional mechanism. The ability of the drug to induce expression of genes associated with mature myeloid cells suggest the drug may have the potential to promote differentiation of FLT3 AML cells. Finally, combination of ONO-7475 with Sorafenib proved effective in an in vivo AML xenograft model suggesting that combination of ONO-7475 with a FLT3 inhibitor could be efficacious for therapy of AML patients with FLT3 ITD. DisclosuresYasuhiro:Ono Pharmaceutical: Employment. Tanaka:Ono Pharmaceutical: Employment. Yoshizawa:Ono Pharmaceutical: Employment. Cortes:Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Arog: Research Funding. Andreeff:AstraZeneca: Research Funding.

The RNA Binding Protein MSI2 Has Increased RNA Binding Activity in Leukemic Stem Cells Compared to Normal Hematopoietic Stem Cells

The RNA binding protein (RBP) MSI2 has been demonstrated to regulate the self-renewal activity and differentiation program of normal hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells (LSCs) (Kharas et al, Nature Medicine 2010, Park et al, 2013 Journal of Experimental Medicine, Park et al JCI 2015). Yet, the extent of MSI2 targets and their differential activity in these rare cells are not known. Standard techniques for identifying RBP targets, such as RNA-IP and CLIP, require large cell numbers (>10 million) and are technically challenging. Here we employed HyperTRIBE, a recently discovered method (McMahon et al. 2016 and Xu et al., 2017), to globally map the MSI2 targeting network in HSPCs and LSCs. In HyperTRIBE, the catalytic domain of the Drosophila ADAR (adenosine deaminase enzyme) is fused with an RBP. This fusion protein leaves a “fingerprint” on the RBP RNA targets by marking the binding sites with an editing A-to-G event. HyperTRIBE was originally developed in Drosophila, but it is not known whether it can be used as a tool for mapping RBP targets in mammalian systems.To test this HyperTRIBE method, we first overexpressed MSI2-HyperTRIBE fusion, MSI2-ADAR dead catalytic mutant fusion control, or an empty vector control in the human AML cell line MOLM-13. Of the MSI2-HyperTRIBE identified targets, 75% overlapped with the iCLIP targets found in the NB4 cell line, suggesting that HyperTRIBE consistently identifies previously published direct MSI2 targets. We next employed MSI2-HyperTRIBE to determine MSI2 RNA targets in rare hematopoietic stem and progenitor cells. We overexpressed MSI2-HyperTRIBE or controls in murine LSKs in vivo for seven weeks and sorted long term HSCs (LT), short term HSCs (ST), multipotent progenitors (MPP2 and MPP4) for RNA-seq. Using 360 to 20,000 sorted cells and as low as 0.1ng of RNA, we were able to identify MSI2 targets in all four HSPC populations (856 in LT-HSC, 783 in ST-HSC, 659 in MPP2, and 661 in MPP4) for the first time. Homer de novo motif search revealed the previously established MSI2 binding motif in all four HSPC populations. Overlapping the targets in LT-HSC, ST-HSC, and MPPs uncovered the unique MSI2 targeting network in LT-HSCs. Gene set enrichment analysis by ENRICHR demonstrated that these targets have a strong signature matching with the hematopoietic stem cell and myelodysplastic syndrome gene sets. These data can be used to unravel the changes in MSI2 targeting program in different stages of stem and progenitor differentiation for the first time.To decipher the differential regulation of MSI2 in normal HSPCs and LSCs, we performed HyperTRIBE in normal mouse LSKs and MLL-AF9 LSCs in vitro (RNA-sequencing was performed 48 hours after of transduction). Strikingly, there is a higher number of editing sites and target genes in LSCs compared to LSKs (2.5 times more editing sites and 1.4 times more genes) despite lower MSI2-HyperTRIBE expression in LSCs compared to LSKs and comparable endogenous MSI2 expression in both cell types. Overlapping the targets in these two cell types showed 82% of LSK targets were also found in LSCs. Furthermore, LSCs have three times more unique targets than LSKs. We observed, in most of the cases, an increase in the number of binding sites per MSI2 target in LSCs compared to LSKs, suggesting a higher affinity of MSI2 to its mRNA targets in LSCs. Of note, this phenomenon seems to be independent from the target mRNA abundance. Previously established targets such as Hoxa9 and Ikzf2 were found to be more efficiently edited and had more binding sites, indicating that MSI2 was more active in LSCs compared to LSKs. In summary, we demonstrated that MSI2 has higher activity at these genes in LSCs compared to LSKs. These data provide evidence for why leukemia cells are more dependent on MSI2 than HSPCs and suggest that expression of RBPs themselves can be distinct from their targeting activity. Moreover, our strategy provides a new platform for discovery of RBP targets in rare cells that can be generally applied to a wide range of dysregulated RBPs in cancer. DisclosuresNo relevant conflicts of interest to declare.

Chloroquine Derivative Lys05 Overcomes Hypoxia-Induced Chemoresistance in Acute Myeloid Leukemia through Metabolic Disruption

Background: Despite initial responses to standard chemotherapy, most patients with acute myeloid leukemia (AML) relapse and have poor prognoses after subsequent therapy. The hypoxic bone marrow (BM) microenvironment is hypothesized to contribute to clinical resistance through the sheltering of AML blasts and leukemia stem cells (LSCs), allowing them to evade chemotherapy and reinitiate the disease. We have previously shown that AML cell lines cultured under prolonged hypoxia (1% O2) can upregulate autophagy and become resistant to cytarabine (AraC). Furthermore, the inhibition of autophagosome turnover with either Bafilomycin A1 (Baf A1) or chloroquine (CQ) can re-sensitize AML cells to AraC. Additionally, Baf A1 therapy effectively eradicated in vivo functional LSCs in a primary human AML xenotransplantation mouse model. However, clinical development of BafA1 and CQ has been limited by poor drug pharmokinetics. Lys05 is a novel water-soluble CQ derivative which exhibits 10 fold more potent autophagy inhibitory properties than CQ in vitro and is well tolerated in mouse models. Here, we tested the effects of Lys05 on human AML growth, chemoresistance under hypoxia, and in vivo leukemia burden in a human AML xenograft model. We also explored the effects of autophagosome accumulation on cellular metabolism as a new mechanism for targeting chemoresistant AML blasts and LSCs.Methods: Human AML cell line MOLM13 and CD34 positive AML patient samples were cultured under hypoxia (1% O2) or normoxia (21% O2) with cytarabine (AraC) and late-stage autophagy inhibitor Lys05 alone and in combination. After 72 hours of drug exposure, apoptosis was measured using Annexin V/propidium iodide flow cytometry. Metabolic assays were performed using a Seahorse XFe96 analyzer to measure oxygen consumption and extracellular acidification rates using the Mitochondrial or Glycolytic Stress Tests on AML cells treated with late-stage autophagy inhibitors for 48 hours. Mitochondrial mass was measured following staining with MITO-ID Green Detection Kit using flow cytometry. Human xenograft models were performed using NSG mice inoculated with MOLM13 BLIV, a luciferase-expressing human AML cell line, and treated with either vehicle or Lys05 (20 mg/kg 4 days on/3 off for 4 weeks). Tumor burden was assessed using bioluminescence and analyzed with IVIS Living Image Software.Results: Treatment of AML MOLM13 cells with Lys05 in vitro resulted in a dose-dependent decrease in cell proliferation and increased apoptosis. Lys05 treatment further displayed similar results to BafA1 and CQ, enhancing the anti-leukemic effects of AraC and overcoming hypoxia-induced chemoresistance. Seahorse XFe analysis revealed that BafA1 inhibited both basal and maximal mitochondrial respiration under both normoxic and hypoxic conditions at 24 hours. CQ and Lys05, however, had no effect on oxidative phosphorylation under normoxia, yet significantly decreased mitochondrial function under hypoxia. Interestingly, reduced function did not coincide with a decrease in mitochondrial mass, but rather increased mitochondrial mass. We also observed decreases in glycolytic function after treatment with late-stage autophagy inhibitors under hypoxia suggesting these cells may be undergoing an energy crisis. Treatment of leukemia-bearing mice with Lys05 trended toward decreased tumor burden compared to vehicle. Studies optimizing Lys05 monotherapy and testing combination treatment with AraC are underway.Conclusions: Our research shows that CQ derivative Lys05 exhibits significant anti-leukemic activity against human AML cells and restores chemosensitivity to cytarabine under hypoxia, further supporting our hypothesis that autophagosome accumulation can overcome hypoxia-induced chemoresistance. We also demonstrate that Lys05 may effectively inhibit leukemia progression in vivo. Our studies further reveal metabolic disruption through the accumulation of autophagosomes as a mechanism for restoring chemosensitivity of AML cells under hypoxia and targeting LSCs. These results establish late-stage autophagy inhibitors, specifically Lys05, as a promising new treatment approach for resistant AML and supports further study of these drugs for treatment of minimal residual disease. [Display omitted] DisclosuresGuzman:Cellectis: Research Funding. Wang:Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Consultancy; Jazz: Speakers Bureau.

SHP-1 As a Negative Regulator of FLT3 Associated Aberrant Signaling and a New Potential Target to FLT3-ITD Mutation

Background: FLT3-ITD mutation remains a great challenge in treating acute myeloid leukemia (AML) patients, for the high incidence of early relapse and drug-resistant mutations to tyrosine kinase inhibitors (TKIs). The FLT3-ITD mutation results in a significant and sustained activation of downstream signaling pathways. However, the physiological negative regulators of those aberrantly activated pathways have not been fully elucidated. In this study, we aim to identify and exploit a key molecule directly interacting with FLT3 receptor and down-regulating FLT3 associated pathways.Methods: The potential proteins interacting with FLT3 were immunoprecipitated (IP) and screened by mass spectrometry. The binding of phosphatase SHP-1 and FLT3 was verified using co-IP and in situ proximity ligation assay (PLA) in cell lines and primary samples. The phosphatase activity and phosphorylation level of SHP-1 was determined by pNPP phosphatase activity assay and western blot. Biological effects of overexpressed SHP-1 by lentiviral vectors in AML cell-lines were assessed using proliferation, apoptosis and clonal formation assays in vitro and xenografts models in vivo. Plasmids of truncated or mutated structure of SHP-1 were constructed and overexpressed in cells to identify the key domain responsible for binding with FLT3 receptor. An artificial trans-membrane peptide was designed to activate SHP-1 activity and promote apoptosis in AML cells.Results: The researchers performed immunoprecipitations (IP) followed by LC-MS/MS based screen in human leukemia cell line MV4-11 for three times, and found that the phosphatase SHP-1 binds to FLT3. In addition, the interaction between SHP-1 and FLT3-WT or FLT3-ITD was verified by co-IP and in situ proximity ligation assay (PLA) in AML cell lines, primary samples and overexpression model of 293-FT.Partial results are showed in Figure A. Furthermore, in heterozygous cell line molm13 and engineered FLT3-ITD knock-in cell line SKM-1 by crispr/cas9 techonology, combined with PLA, pNPP phosphatase activity assay and western blot, we found that SHP-1 may bind with p-FLT3 and acquired phosphatase activity(Figure B-D). In turn, p-FLT3 itself and some important proteins in the downstream pathway would be dephosphorylated and the signal transmission would be terminated. In FLT3-ITD cells, the phosphorylation level of SHP-1 significantly reduced.By using lentivirus to overexpress or knockdown shp-1 protein in FLT3-WT and FLT3-ITD cells, the biological effects were studied. Experiments on cell apoptosis, proliferation and clonal formation have shown that overexpression of SHP-1 in AML cells may promote apoptosis, inhibit proliferation and clone formation, especially in FLT3-ITD cells. On the contrary, inhibition of shp-1 could promote proliferation and increase the ability of clone formation. Furthermore, DOX-inducible overexpression cell line of SHP-1 was obtained by tet-on virus system and the western blots showed that SHP-1 may inhibit p-ERK and p-STAT5 activity. In vivo experiments of NCG mice showed that, the degree of leukemia infiltration in peripheral blood was decreased in AML transplantation mice induced by DOX to express SHP-1 when compared to control group.Next, according to the analysis of protein structure by computer simulation, we constructed truncated and mutated plasmids and overexpressed in cells, then using PLA to verify the binding , we found that FLT3 interact with N-SH2 domain of SHP-1,exactly the R30 site(Figure E). Therefore, by using specific trans-membrane peptide, the researchers found that it could activate SHP-1 and promote apoptosis in AML cells, especially FLT3-ITD ones, inhibit cell proliferation and the downstream signal pathway of FLT3.Lastly,injection of peptide in subcutaneous tumor model of nude mouse showed that the tumor was significantly smaller than that of the control group(Figure F).Conclusion: We identify a negative regulator, namely phosphatase SHP-1, which directly interacts with FLT3 receptor through the N-SH2 domain and down-regulates FLT3 downstream signaling pathways. Overexpression of SHP-1 promotes apoptosis and inhibits proliferation in cells with FLT3-ITD mutation. Artificial trans-membrane peptides activating SHP-1 can reverse FLT3 associated aberrant signaling, thus providing a new potential strategy to target FLT3-ITD in AML. The possible mechanism is showed in figure G. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MLL-Fusion Leukemia Dependence on MBNL1 Is Associated with Alternative Splicing of Oncogenic Proteins

Leukemia is the most common childhood cancer, and while outcomes for most children have improved significantly, the prognosis in infant leukemia remains dire. The majority of infant leukemia, either acute myeloid (AML) or acute lymphoid (ALL), is caused by reciprocal translocations of the MLL-gene. Prior studies show that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is the RNA binding protein muscleblind-like 1 (MBNL1). We found that MBNL1 knockdown significantly impairs propagation of MLL-rearranged (MLLr) leukemic cells in vitro and in vivo using human cell lines and transformed murine cells. To further characterize the role of MBNL1 in acute leukemia, we performed shRNA knockdown experiments in MLLr and non-MLLr leukemia cell lines and in primary patient samples. While MBNL1 knockdown does also impair growth of non-MLLr leukemic cells, the effect is less pronounced. In a 5-day growth experiment MBNL1-knockdown MLLr cells (THP-1) displayed a median 71% reduced growth compared to controls, whereas non-MLLr cells (HL-60) displayed only a median 32% growth reduction (p=0.0001). Cells from two patients with MLLr AML (one with MLL-AF9 and one with MLL-AF10 fusion) underwent shNT (non-targeting) or shMBNL1 transduction.. Unsorted cells were transplanted into NSGS mice. Mice were observed until showing signs of distress and then analyzed for engraftment of human cells and abundance of transduced cells (venus-positive). In the shNT group there was robust persistence of transduced cells (7%-98% of human cells), whereas shMBNL1-transduced cells were not detected or comprised <1% of human cells in most of the recipient mice.Given that MBNL1 is known to regulate alternative splicing, we used unbiased RNAseq along with a novel analytic splice-junction and intron-quantification toolkit (AltAnalyze) to determine splicing changes induced by knockdown of MBNL1 in the MLLr leukemia cell line MOLM-13. In a parallel analysis, we determined splicing differences between MLLr and cytogenetically-normal (CN) AML patient samples. We then compared these two results to determine the splicing events regulated by MBNL1 and assess the contribution of MBNL1 to splicing events observed in primary MLLr leukemias. Strikingly, this comparative analysis found that 88% of overlapping differentially expressed splicing events (75 out of 85) were concordant between patient MLLr and CN-AML as compared to control versus MBNL1 knockdown. The most common class of splicing event that occurred with MBNL1 knockdown was intron retention. Specifically, our findings suggest that MBNL1 knockdown restores intron retention, and that MBNL1 overexpression promotes expression of protein-coding genes that would otherwise be suppressed through intron retention-introduced premature termination codons. Several genes whose transcripts are alternatively spliced by MBNL1 have prior associations with cancer, most notably DOT1L and SETD1A which are specifically implicated in MLLr leukemia. Splicing validation through RT-PCR confirmed increased intron retention in DOT1L and SETD1A transcripts after MBNL1 knockdown. Interestingly, one target of MBNL1 is the MBNL1 mRNA itself, with resultant exclusion of exon 5. MBNL1 lacking exon 5 has a stronger affinity to RNA. In summary, our data suggests that MBNL1 plays a key role in the pathogenesis of MLL-fusion leukemia, wherein it stabilizes the transcripts of multiple leukemogenic genes including DOT1L and SETD1A. Proteins such as DOT1L are critical for transcriptional activation of downstream targets of the MLL-fusion protein (including activation of MBNL1, creating a positive feedback loop). Additionally, high levels of MBNL1 protein may alter splicing in ways that enhance MBNL1 functionality. DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide and Sorafenib Induce Synthetic Lethality of FLT3-ITD Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) with Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation is notoriously hard to treat. We identified two drugs that together form an effective combination therapy against FLT3-ITD AML. One of the drugs, Sorafenib, an inhibitor of FLT3-ITD and other kinase activity, produces an impressive but short-lived remission in FLT3-ITD AML patients. The second, arsenic trioxide (ATO), at therapeutically achievable concentrations, reduces the level of FLT3-ITD and Mcl-1 proteins, and induces apoptosis in leukemic cell lines and in primary cells expressing FLT3-ITD. We linked this relative sensitivity to ATO to low levels of reduced glutathione. While producing proapoptotic effects, ATO treatment also has an unwanted effect whereby it causes the accumulation of the phosphorylated (inactive) form of glycogen synthase kinase 3β (GSK3β), a kinase necessary for apoptosis. When ATO is combined with Sorafenib, GSK3β is activated, Mcl-1 is further reduced, and proapoptotic proteins Bak and Bax are activated. Mice xenografted with FLT3-ITD MOLM13 cell line treated with the Sorafenib/ATO combination have significantly improved survival. This combination has potential to improve the therapeutic outcome of FLT3-ITD-targeted therapy of AML patients. Mol Cancer Ther; 17(9); 1871-80. ©2018 AACR.

N-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Epigenetic modification enhances the cytotoxicity of busulfan and4-hydroperoxycyclophosphamide in AML cells

The combination of the DNA-alkylating agents busulfan (Bu) and cyclophosphamide is the most commonly used myeloablative pretransplantation conditioning therapy for myeloid leukemias. However, it is associated with significant nonrelapse mortality, which prohibits dose escalation to control relapse. We hypothesized that combining these two drugs with an epigenetic modifier would increase antileukemic efficacy without jeopardizing patient safety. A preclinical study was performed to determine the synergistic cytotoxicity of Bu, 4-hydroperoxycyclophosphamide (4HC), and the hypomethylating agent decitabine (DAC) in human acute myeloid leukemia (AML) cell lines. Exposure of KBM3/Bu2506 (P53-null) and OCI-AML3 (P53-wild-type) cells to Bu+4HC inhibited cell proliferation by ∼35-39%; addition of DAC increased the inhibition to ∼60-62%. The observed synergistic interactions correlated with DNA damage response activation, increased the production of reactive oxygen species, and decreased mitochondrial membrane potential, release of mitochondrial proapoptotic proteins into the cytoplasm, and induction of caspase-dependent programmed cell death. The Bu+4HC+DAC combination further caused chromatin trapping of DNMT1 with a concomitant increase in DNA damage. In contrast, FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD)-positive AML cell lines were not sensitized to Bu+4HC by inclusion of DAC; addition of the FLT3 kinase inhibitor sorafenib sensitized the FLT3-ITD-positive MV4-11 and MOLM13 cell lines to the triple drug combination by inhibiting the FLT3 signal transduction pathway. Our results therefore provide a rationale for the development of personalized conditioning therapy for patients with P53-mutated and FLT3-ITD-positive AML.

Discovery of (S)-2-amino-N-(5-(6-chloro-5-(3-methylphenylsulfonamido)pyridin-3-yl)-4-methylthiazol-2-yl)-3-methylbutanamide (CHMFL-PI3KD-317) as a potent and selective phosphoinositide 3-kinase delta (PI3Kδ) inhibitor

PI3Kδ, which is mainly expressed in leukocytes, plays a critical role in B-cell receptor mediated signaling pathway and has been extensively studied as a drug discovery target for B cell malignances such as AML, CLL etc. In this manuscript, we report the discovery, SAR optimization and pharmacological evaluation of a novel series of aminothiazole-pyridine containing PI3Kδ inhibitors. Among them compound 15i (CHMFL-PI3KD-317) displays an IC50 of 6 nM against PI3Kδ in the ADP-Glo biochemical assays. It also exhibits over 10–1500 fold selectivity over other class I, II and III PIKK family isoforms. In addition, in the cellular context, 15i can selectively and potently inhibit PI3Kδ mediated phosphorylation of Akt T308 but not PI3Kα, β, γ mediated Akt phosphorylation. 15i also exhibits an excellent selectivity profile in the protein kinases including 468 kinases/mutants at the concentration of 1 μM. 15i has acceptable pharmacokinetic properties and can dose-dependently inhibit the tumor growth of AML cell line MOLM14 inoculated xenograft mouse model. The high selectivity and potency makes 15i a potential valuable addition to the current PI3Kδ armory.

Selective DOT1L, LSD1, and HDAC Class I Inhibitors Reduce HOXA9 Expression in MLL-AF9 Rearranged Leukemia Cells, But Dysregulate the Expression of Many Histone-Modifying Enzymes.

Mixed lineage leukemia results from chromosomal rearrangements of the gene mixed lineage leukemia (MLL). MLL-AF9 is one such rearrangement that recruits the lysine methyltransferase, human disruptor of telomere silencing 1-like (DOT1L) and lysine specific demethylase 1 (LSD1), resulting in elevated expression of the Homeobox protein A9 (HOXA9), and leukemia. Inhibitors of LSD1 or DOT1L reduce HOXA9 expression, kill MLL-rearranged cells, and may treat leukemia. To quantify their effects on histone modifying enzyme activity and expression in MLL-rearranged leukemia, we tested inhibitors of DOT1L (EPZ-5676), LSD1 (GSK2879552), and HDAC (mocetinostat), in the MLL-AF9 cell line MOLM-13. All inhibitors reduced MOLM-13 viability but only mocetinostat induced apoptosis. EPZ-5676 increased total histone lysine dimethylation, which was attributed to a reduction in LSD1 expression, and was indistinguishable from direct LSD1 inhibition by GSK2879552. All compounds directly inhibit, or reduce the expression of, HOXA9, DOT1L and LSD1 by qPCR, increase total histone lysine methylation and acetylation by LC-MS/MS, and specifically reduce H3K79Me2 and increase H3K14Ac. Each inhibitor altered the expression of many histone modifying enzymes which may precipitate additional changes in expression. To the extent that this decreases HOXA9 expression it benefits mixed lineage leukemia treatment, all other expression changes are off-target effects.

Abstract 804: Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia

Abstract Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Activating mutations of internal tandem duplication (ITD) and tyrosine kinase domain point mutations (TKD) of FLT3 have been reported in approximately 30% of AML as oncogenic driver mutations. Currently, FLT3 inhibitors showed clinical benefits in the corresponding AML patients. In this study, we have characterized a novel FLT3 inhibitor, HM43239, and assessed the potential as a novel therapy for AML patients. Materials and Methods: HM43239 is a novel, highly potent FLT3 kinase inhibitor. This compound showed tight binding to FLT3 kinase in in silico docking model as a reversible Type I inhibitor. In vitro kinase assay was performed to identify kinase selectivity of HM43239. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML cell lines. HM43239 was evaluated in MV-4-11 and MOLM-13 xenograft mice models. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: Among 191 kinases biochemically assayed, HM43239 showed the high selectivity toward FLT3 and AML associated other kinases (e.g. SYK, JAK and TAK1). IC50s' of HM43239 against FLT3 WT, FLT3 ITD and FLT3 D835Y kinases were 1.1 nM, 1.8 nM and 1.0 nM, respectively. HM43239 potently inhibited the growth of AML cell lines harboring FLT3 ITD mutation, such as MV4-11 (IC50: 1.3 nM), MOLM-13 (5.1 nM) and MOLM-14 (2.9 nM). Furthermore, HM43239 effectively inhibited the phosphorylation levels of FLT3 and of downstream kinases related with cell proliferation. In addition, caspase 3/7-dependent apoptosis was induced by HM43239 in AML cell lines expressing FLT3 ITD mutant. And HM43239 inhibited proliferation and induced apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells) suggesting that the possibility for targeting LSC. HM43239 showed the excellent dose proportional antitumor activity in mouse models xenografted with both MV4-11 and MOLM-13 cell line without any significant toxicity. Moreover, we identified in vivo modulation of related targets (p-FLT3 &amp; p-STAT5) in AML cell with FLT3 mutant. Conclusion: Taken together, HM43239 has demonstrated the potential therapeutic efficacy for the treatment of AML patients and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: Miyoung Lee, Young Eun Ha, Mi Jin Moon, Joo-Yun Byun, HyunKyung Yu, SeokJong Kang, JaeHo Lee, Kyuhang Lee, Eunkyung Kim, Eunyoung Kim, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in 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 804.

Abstract 4522: An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples

Abstract FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [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 4522.

Abstract 4821: A targeted differentiation therapy for the treatment of acute myeloid leukemia

Abstract Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities present in all ages, but mainly prevalent in an elderly population with an average age of above 60 years. The treatment of elderly AML remains a formidable challenge as the long-term outcomes of elderly AML patients have not improved in the last three decades, with ≤5% overall survival recorded at 5 years, calling for novel treatment options. Epigenetic therapy has a significant impact on the management of hematologic malignancies. Recent findings show that using very low dosages of decitabine depletes DNA methyl transferase 1 (DNMT1) without cytotoxicity. Decitabine-mediated epigenetic therapy does not induce FLT3 (fms-like tyrosine kinase 3) ligand, which hinders the effectiveness of FLT3 inhibitors. The inhibition of FLT3 and DNMT1 is associated with terminal myeloid differentiation of AML cells. Therefore, combining the inhibitors of FLT3 and DNMT1 may be an effective therapeutic approach for the treatment of poor-risk AML. Ponatinib is a third-generation receptor tyrosine kinase inhibitor. Various in vitro and in vivo preclinical studies demonstrated antileukemic activities of ponatinib against AML cells bearing FLT3-ITD mutations. Methods: Apoptosis and CD11b were measured by flow cytometry. FLT3 signaling and DNMT1 levels were analyzed by immunoblotting. Results: FLT3-ITD expressing AML cell lines MV4-11, MOLM-13, and MOLM-14 were used in the study. Co-treatment with decitabine (10-100 nM) and ponatinib (2 nM) in AML cells induced apoptosis in association with PARP cleavage. Increased levels of pro-apoptotic protein BAD and decreased levels of anti-apoptotic protein MCL1 were observed in these treated cells. The combination of decitabine and ponatinib also showed similar effects in primary AML cells expressing FLT3-ITD. The Western blot analysis demonstrated that treatment of decitabine decreased levels of DNMT1 and ponatinib inhibited FLT3 signaling and activation of downstream effectors STAT5, AKT, and ERK1/2 with induction of PU.1 a key regulator of myeloid differentiation. Co-treatment with ponatinib and decitabine using low concentrations in MV4-11 and MOLM14 cells induced myeloid differentiation. The percentage of differentiated cells was measured by increased surface expression of CD11b analyzed by flow cytometry and granulocytic/monocytic morphology examined by Wright-Giemsa staining. Conclusions: Mechanistically, the hypomethylating agent initiates the differentiation process and FLT3 inhibition augments differentiation leading to apoptosis of AML cells. Altogether, these preclinical findings of downregulation of DNMT1 and induction of PU.1 are a novel differentiation approach to induce apoptosis in AML cells and warrant future therapeutic potential for the treatment of AML patients expressing FLT3 mutations. Citation Format: Sudhakiranmayi Kuravi, Myles Taylor, Tara L. Lin, Jensen Roy, Joseph McGuirk, Ramesh Balusu. A targeted differentiation 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 4821.

Abstract 805: NMS-P088, a FLT3-KIT-CSF-1R inhibitor with activity on FLT3 F691L as a novel agent in AML

Abstract FLT3, KIT and CSF1R are members of the class III receptor tyrosine kinase family, characterized by an autoinhibitory juxtamembrane (JM) domain that docks within the kinase domain to stabilize a catalytically inactive conformation. Activating rearrangements of the JM domain of FLT3 (FLT3-ITD) occur in 20-25% of AML and represent a driver of disease and a negative prognostic factor. Another 5-7% of AML harbours an activating D835 mutation in the activation loop of the kinase domain. Several FLT3 inhibitors, including quizartinib, crenolanib and gilteritinib, are currently in advanced clinical testing. Moreover midostaurin, a multikinase inhibitor with activity on FLT3, has been recently approved in FLT3 mut AML in combination with standard chemotherapy. Unfortunately, the clinical response to FLT3 inhibitors is shortened by the emergence of resistance mutations at critical residues such as D835 and F691, the so called “gatekeeper” residue. F961L mutations have been described to induce resistance to quizartinib, crenolanib and gilteritinib, making F691L an unmet medical need in AML. NMS-P088 is a potent and selective inhibitor of FLT3 and KIT kinases, including variants with both primary and secondary resistance mutations, as well as of CSF1R, with exquisite cellular selectivity for cell lines dependent on these targets. NMS-P088 has remarkable (subnanomolar) activity against the MOLM-13 and MV4-11 AML cell lines harbouring the FLT3-ITD rearrangement. On a panel of BA/F3 cells harbouring FLT3-ITD and its mutant forms NMS-P088 showed high potency and very significant activity on F691L, superior to that of key comparators tested in parallel. In the disseminated MOLM-13 AML model, repeated oral administration of NMS-P088 as single agent was able to significantly increase survival time, and showed synergy with cytarabine. The outstanding in vitro activity of NMS-P088 on BA/F3_FLT3-ITD harbouring the F691L mutation was also confirmed in vivo, with high TGI for NMS-P088, while quizartinib tested in parallel resulted not active. NMS-P088 has activity on CSF1-dependent macrophages both in vitro and in vivo and showed single agent efficacy in a syngeneic tumor model done in immunocompetent mice, with robust reduction of CSF-1R positive intratumoral macrophages, providing a rationale for testing in clinical setting as a modulator of host vs tumor response. GLP toxicity studies revealed good tolerability at efficacious exposures, with no cardiac effects and excellent BBB penetration. Thus NMS-P088, a potent FLT3 inhibitor with activity on the gatekeeper mutation, is a preclinical candidate with potential to address an unmet medical need in AML, both as single agent and in combination, as well as for testing in different solid tumors sensitive to immunomodulation. Citation Format: Marina Ciomei, Elena Ardini, Gemma Texido, Rachele Alzani, Wilma Pastori, Dario Ballinari, Sabrina Cribioli, Fabio Gasparri, Nilla Avanzi, Daniele Casero, Daniele Donati, Arturo Galvani, Andrea Lombardi Borgia, Antonella Isacchi. NMS-P088, a FLT3-KIT-CSF-1R inhibitor with activity on FLT3 F691L as a novel agent in AML [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 805.

Abstract 1890: Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38- cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38+ cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38- HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38+ cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38- cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38- AML cell lines and the density of CD38 increased at the surface of CD38+ AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab. Citation Format: Enguerran Mouly, Cécile Planquette, Emilie Rousseau, Rémi Delansorne. Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6 [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 1890.

Abstract 883: Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92

Abstract The positive-transcription elongation factor (P-TEFb) complex is critical in stimulating the transcription of non-abortive transcripts by RNA polymerase II. The CDK9/cyclin-T heterodimer comprises the P-TEFb complex and promotes transcription elongation through phosphorylation of serine 2 of the heptapeptide repeats of the c-terminal domain of RNA Polymerase II (RNAPII-CTD). Alvocidib, a potent CDK9 inhibitor, has been shown to suppress expression of MCL-1 and Myc mRNAs, and clinical pharmacodynamic data suggest that alvocidib's activity is primarily mediated through the suppression of MCL-1 mRNA and protein expression. In addition to MCL-1 suppression, we hypothesized that CDK9 inhibition affects other RNA Polymerase II target RNAs, such as microRNAs. Recently, it was reported that inhibition of bromodomain and extraterminal domain (BET) protein by JQ1 suppresses miR17-92 expression. miR17-92 negatively regulates expression of the pro-apoptotic BH3-only protein, BIM, leading to suppression of BIM expression, thereby decreasing the cells' ability to induce apoptosis. It was hypothesized that CDK9 kinase activity is key to miR17-92 expression and suppression of BIM expression and that targeting CDK9 would lead to a decrease in miR17-92, and increases in BIM. MV4-11, OCI-AML3, MOLM13, and THP1 AML cell lines were used to determine the effects of alvocidib treatment on microRNAs. RT-qPCR was utilized to determine microRNA expression levels of miR17-92 and mRNA levels of BIM and other markers. Protein changes were determined using standard gel electrophoresis and immunoblotting technique. CellTiter-Glo and Caspase-Glo were used for all cell viability and apoptosis assays interrogating alvocidib. With CDK9 inhibition mediated via alvocidib, a dose- and time-dependent decrease in miR17-92 expression in MV4-11, OCI-AML3, MOLM13, and THP1 cells was observed. A decrease was observed even 3 hours post-treatment, persisting for up to 24 hours. MicroRNA suppression following treatment ranged in magnitude, with a maximal effect of between 3.3 to 4.2-fold suppression, depending on the cell line and timepoint. This suppression coincided with an increase in BIM mRNA and protein expression in the MV4-11, OCI-AML3, and MOLM13 cells. However, BIM protein increase was not observed in THP1 cells. Maximal increase in BIM mRNA levels reached 5.4-fold in the MV4-11 cell line. Our data suggest that CDK9 inhibition suppresses RNA Polymerase II-mediated expression of miR17-92, which in turn leads to increased expression of BIM. Combined with MCL-1 reduction, increased BIM protein expression mediated by alvocidib leads to enhanced apoptosis. Taken together, the data provide additional understanding of CDK9 as a potential therapeutic target, and are consistent with the hypothesis that CDK9 activity is necessary for miR17-92 expression. Citation Format: Hillary Haws, Hubert F. Arokium, James L. Bogenberger, Adam Siddiqui-Jain, David J. Bearss, Raoul Tibes, Steven L. Warner, Clifford J. Whatcott. Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92 [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 883.