Therapeutic Strategy For Acute Myeloid Leukemia Research Articles

Direct Pharmacological Inhibition of the Transcription Factor PU.1 in Acute Myeloid Leukemia

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells.We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments.The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition.To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers.Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival.To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. DisclosuresWill:Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl:Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

NADPH oxidase-2 derived superoxide drives mitochondrial transfer from bone marrow stromal cells to leukemic blasts

AbstractImprovements in the understanding of the metabolic cross-talk between cancer and its microenvironment are expected to lead to novel therapeutic approaches. Acute myeloid leukemia (AML) cells have increased mitochondria compared with nonmalignant CD34+ hematopoietic progenitor cells. Furthermore, contrary to the Warburg hypothesis, AML relies on oxidative phosphorylation to generate adenosine triphosphate. Here we report that in human AML, NOX2 generates superoxide, which stimulates bone marrow stromal cells (BMSC) to AML blast transfer of mitochondria through AML-derived tunneling nanotubes. Moreover, inhibition of NOX2 was able to prevent mitochondrial transfer, increase AML apoptosis, and improve NSG AML mouse survival. Although mitochondrial transfer from BMSC to nonmalignant CD34+ cells occurs in response to oxidative stress, NOX2 inhibition had no detectable effect on nonmalignant CD34+ cell survival. Taken together, we identify tumor-specific dependence on NOX2-driven mitochondrial transfer as a novel therapeutic strategy in AML.

Abstract 3340: Identification of CARM1/PRMT4 as a novel therapeutic target for AML

Abstract Chromatin modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs) have emerged as important targets in cancer. PRMT4, also known as CARM1, is overexpressed in a number of cancers, including breast, prostate, pancreatic, and lung cancer. Our lab reported the overexpression of PRMT4 in the context of acute myeloid leukemia (AML), showing that more than 70% of cytogenetically normal AML patients have up-regulation of PRMT4. Here, we investigated the role of PRMT4 in normal hematopoiesis and leukemia development. In order to study the role of PRMT4 in normal hematopoiesis, Prmt4-floxed mice were crossed with Vav1-cre mice purchased from the Jackson Laboratory. Inducible Prmt4 conditional KO mice were generated by crossing Prmt4-floxed mice with Mx1-Cre mice and Prmt4 gene excision was induced by poly(I:C). Using this hematopoietic specific knockout system, we show that loss of PRMT4 has little effect on normal hematopoiesis, but promotes the differentiation of hematopoietic stem and progenitor cells. Next we evaluated the role of PRMT4 in leukemia development using leukemia transplantation models driven by fusion oncoproteins. Strikingly, the knockout of PRMT4 completely abrogates leukemia initiation in fetal liver transplantation models driven by the AML1-ETO or MLL-AF9 fusion proteins. We further characterized the mechanism for the leukemia-specific dependence on PRMT4 using leukemia cell lines and found that knockdown of PRMT4 impairs cell cycle progression, decreases proliferation, and induces rapid apoptosis. To examine PRMT4 dependent changes in gene expression in a leukemia system, we used lentiviral vectors that express RFP and shRNAs directed against PRMT4. We knocked down PRMT4 in four leukemia cell lines or normal human cord-blood derived CD34+ cells. Gene set enrichment analysis showed that all four leukemia cell lines with knockdown of PRMT4 significantly down-regulated E2F target genes compared to the scrambled control. Chromatin immunoprecipitation analysis (ChIP) confirmed the presence of PRMT4 and H3R17 dimethylation at the promoter regions of E2F1 targets. The PRMT4 conditional knockout mice and PRMT4 knockdown experiments both suggest that the loss of PRMT4 protein has a selective effect on leukemia cells compared to normal hematopoietic stem and progenitor cells. Collectively, this work suggests that targeting PRMT4 through chemical inhibition may be an effective therapeutic strategy for AML and other cancers with up-regulation of PRMT4. Citation Format: Sarah M. Greenblatt, Pierre-Jacques J. Hamard, Takashi Asai, Na Man, Concepcion Martinez-Caja, Fan Liu, Stephen Nimer. Identification of CARM1/PRMT4 as a novel therapeutic target for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2017-3340

Retinoic Acid-induced differentiation sensitizes myeloid progenitors cells to ER stress

The clonal expansion of hematopoietic myeloid precursors blocked at different stages of differentiation characterizes the acute myeloid leukemia (AML) phenotype. A subtype of AML, acute promyelocytic leukemia (APL), characterized by the chimeric protein PML-RARα is considered a paradigm of differentiation therapy. In this leukemia subtype the all-trans-retinoic acid (RA)-based treatments are able to induce PML-RARα degradation and leukemic blast terminal differentiation [1-2]. Granulocytic differentiation of APL cells driven by RA triggers a physiological Unfolded Protein Response (UPR), a series of pathways emanating from the ER in case of ER stress, which ensues when higher protein folding activity is required as during differentiation. We show here that, although mild, the ER stress induced by RA is sufficient to render human APL cell lines and primary blasts very sensitive to low doses of Tunicamycin (Tm), an ER stress inducing drug, at doses that are not toxic in the absence of RA. Importantly only human progenitors cells derived from APL patients resulted sensitive to the combined treatment with RA and Tm whereas those obtained from healthy donors were not affected. We also show that the UPR pathway downstream of PERK plays a major protective role against ER stress in differentiating cells and, by using a specific PERK inhibitor, we potentiated the toxic effect of the combination of RA and Tm. In conclusion, our findings identify the ER stress-related pathways as potential targets in the search for novel therapeutic strategies in AML.

Inhibition of ATR in Combination with Nucleoside Analogues Eradicates Acute Myeloid Leukaemia in an Orthotopic Murine Xenograft Model

The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts.Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine.The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo.We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p<0.001). Nevertheless, leukemic cells persisted and the cause of death in all mice randomised to this arm was relapsed disseminated disease. Treatment with gemcitabine and VX-970 in combination also conferred very significant early disease control (p=0.002), which eradicated luciferase signal by day 21 in all 6 mice randomised to this arm (Figure 1), and also conferred significantly improved overall survival compared to controls (p<0.001) or mice treated with gemcitabine monotherapy (p=0.001), with four mice remaining disease free at day 126. No significant weight loss or toxicity was observed in any of the treatment arms. Taken together, these data suggest ATR inhibition as a promising therapeutic strategy in AML. [Display omitted] DisclosuresPollard:Vertex Pharmaceuticals (Europe) Ltd.: Employment.

Pacritinib Targets IRAK1 and Shows Synergy with HDAC and BET Inhibitors in Acute Myeloid Leukemia

Background: The bone marrow microenvironment modulates response of acute myeloid leukemia (AML) to therapy. Thus necessitating the identification of microenvironment-driven signaling mechanisms that contribute to AML pathogenesis. We recently showed that AML growth depends on IL-1 signaling in a majority of cases that include a spectrum of genetic aberrations. IL-1 signals through the serine/threonine kinase IL-1 receptor-associated kinase (IRAK1), a central mediator of immune and inflammatory responses that was recently suggested as a potential therapeutic target in leukemia. Pacritinib is currently under development as a small-molecule JAK2 inhibitor for myelofibrosis; recently a kinome screen of 429 kinases showed that pacritinib also inhibits IRAK1 activity (IC50 = 13.6 nM). Here we provide further evidence to support targeting of IRAK1 by pacritinib as a therapeutic strategy in AML, and show synergistic activity with histone deacetylase (HDAC) or bromodomain (BET) inhibitors to overcome drug resistance across genetic subtypes.Methods: We used inducible shRNA to reduce IRAK1 levels in primary AML cells. To validate the selectivity of pacritinib for IRAK1, we performed targeted mutagenesis on key interacting residues. We compared the efficacy of pacritinib, an established dual FLT3/JAK2 inhibitor, to that of the FLT3 inhibitor quizartinib and the JAK2 inhibitor ruxolitinib in 12 cell lines and 46 primary AML samples with various genetic lesions. We tested the synergy of pacritinib in combination with various drugs, including HDAC, BET, and BTK inhibitors, and BH3 mimetic. Equimolar concentrations of both agents were tested at a dose gradient of 1.0-5000 nM. Synergy and combination index were calculated using Calcusyn software. The effects of drug treatments were evaluated on cell viability, survival, differentiation, and downstream signaling.Results: Genetic knockdown of IRAK1 reduced the viability of AML cell lines and primary AML cells by 70% and reduced p38 and ERK1/2 kinase activity. Molecular docking simulation showed that pacritinib forms favorable interactions with D298 and S295 residues in the IRAK1 kinase domain. IRAK1D298K and IRAK1S295D conferred significant pacritinib resistance compared to native IRAK1, confirming the selective binding and on-target inhibitory effect of pacritinib. Primary AML samples and cell lines showed sensitivity to pacritinib (median IC50 = 90 nm, range = 2.0-1000 nM, p<0.004), with a higher response rate (44/46 or 96%, or IC50 < 1000 nM) than that observed with ruxolitinib (15%) or quizartinib (80%). With regard to IC90 < 1000 nM, a more stringent measure of efficacy, a higher percentage of primary AML samples (33%) showed sensitivity to pacritinib than that for quizartinib (13%, p<0.0001). Mechanistic studies showed that pacritinib inhibits the activity of downstream effectors in the IL-1 pathway, including IRAK1 and p38, in addition to inhibiting the secretion of various inflammatory cytokines. Consistent with previous studies, pacritinib inhibited FLT3 and JAK2/JAK3 activity in FLT3- or JAK-dependent AML cells, respectively. Additionally, our data suggest that pacritinib reduced the viability of AML primary samples and cell lines harboring a variety of genetic abnormalities, including TET2, MLL, ASXL1, NPM1, CEBPA, and RAS mutations, by inhibiting IRAK1 activity. To identify potential combination therapies, we combined pacritinib with agents from several classes and found synergy with the HDAC inhibitor panobinostat and the BET inhibitor JQ1 with a combination index <1. Specifically, treatment with 50 nM pacritinib or 50 nM JQ1 alone reduced the viability of primary AML cells to 50%, while the combined treatment reduced viability to ~25%. Similarly, 50 nM pacritinib or 20 nM panobinostat alone reduced the viability of AML cells to 50%, while the combined treatment reduced viability to ~10%. Pacritinib blocked cell growth, while HDAC or BET inhibitors promoted differentiation of AML cells and reduced numbers of early progenitors. The combinations blocked not only IRAK1 and ERK activity, but also c-Myc.Conclusions: IRAK1 is a potential therapeutic target in AML across genetic subtypes. Pacritinib, in addition to suppressing JAK2 and FLT3 signaling, specifically blocks IRAK1 signaling in AML and synergizes with HDAC or BET inhibitors to block growth and promote differentiation, establishing its potential clinical utility. DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Singer:CTI BioPharma Corp.: Employment, Equity Ownership, Other: Leadership . Agarwal:CTI BioPharma Corp: Research Funding.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Targeting prohibitins induces apoptosis in acute myeloid leukemia cells.

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins (PHBs). In this study, the pro-apoptotic effect of fluorizoline was assessed in two cell lines and 21 primary samples from patients with debut of acute myeloid leukemia (AML). Fluorizoline induced apoptosis in AML cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespectively of patients' clinical or genetic features. In addition, fluorizoline inhibited the clonogenic capacity and induced differentiation of AML cells. Fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. These results suggest that targeting PHBs could be a new therapeutic strategy for AML.

Evaluation of artemisinins for the treatment of acute myeloid leukemia.

Investigate antileukemic activity of artemisinins, artesunate (ART), and dihydroartemisinin (DHA), in combination with cytarabine, a key component of acute myeloid leukemia (AML) chemotherapy using in vitro and in vivo models. Using ten human AML cell lines, we conducted a high-throughput screen to identify antimalarial agents with antileukemic activity. We evaluated effects of ART and DHA on cell viability, cytotoxicity, apoptosis, lysosomal integrity, and combination effects with cytarabine in cell lines and primary patient blasts. In vivo pharmacokinetic studies and efficacy of single-agent ART or combination with cytarabine were evaluated in three xenograft models. ART and DHA had the most potent activity in a panel of AML cell lines, with selectivity toward samples harboring MLL rearrangements and FLT3-ITD mutations. Combination of ART or DHA was synergistic with cytarabine. Single-dose ART (120mg/kg) produced human equivalent exposures, but multiple dose daily administration required for in vivo efficacy was not tolerated. Combination treatment produced initial regression, but did not prolong survival in vivo. The pharmacology of artemisinins is problematic and should be considered in designing AML treatment strategies with currently available agents. Artemisinins with improved pharmacokinetic properties may offer therapeutic benefit in combination with conventional therapeutic strategies in AML.

Abstract A04: Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy

Abstract Metabolic alterations are a cancer hallmark that are typically evaluated in bulk tissues. However most normal tissues and cancers are hierarchically organized and the metabolic requirements of both normal and cancer stem cells are poorly understood, particularly beyond energy metabolism. By analyzing a comprehensive transcriptional roadmap of human hematopoiesis, and comparing this to a leukemia stem cell (LSC) signature developed from 84 human acute myeloid leukemia (AML) samples, we found that: (1) several metabolic pathways, specifically in bioactive lipids, distinguish normal hematopoietic stem cells (HSC) from progenitors; and (2) while LSC are similar to HSC, specific metabolic pathways are more comparable to those of normal progenitors. We defined a lipid stem signature of 24 lipid genes, including sphingolipid genes, whose expression is higher in HSC than progenitors. Interestingly, sphingosine-1-phosphate (S1P) is known to play a role in HSC egress, and ceramide vs S1P levels serve as a rheostat to regulate cell growth and survival. To determine if sphingolipids play a functional role in the primitive hematopoietic compartment, we altered sphingolipid signaling by plating sorted populations of HSC or granulocyte-myeloid progenitors (GMP) in methylcellulose containing myriocin, which inhibits the first step of de novo sphingolipid synthesis, or FTY720, a S1P mimetic. Myriocin decreased GMP colony output but did not affect CFC derived from HSC. By contrast FTY720 affected HSC-derived CFC but not those from GMP, suggesting differential sensitivity to sphingolipid pathway inhibition between stem and progenitor cells. In vitro treatment of lineage depleted cord blood (Lin- CB) with myriocin for 8 days limited only myeloid differentiation compared to control treated cells. In contrast, FTY720 treatment reduced levels of immunophenotypic stem cells, erythroid and myeloid cells, as would be expected with inhibition of S1P proliferative signaling. However, in vitro FTY720-treated Lin- CB cells exhibit 16-week engraftment capacity comparable to that of controlled-treated cells, suggesting that any effects on HSC function are reversible after drug withdrawal. Furthermore, in vivo treatment with FTY720 in mice with established CB grafts did not decrease engraftment. Our lipid stem signature is enriched in LSC gene expression profiles from our 84 AML cohort by GSEA analysis, suggesting that differences in lipid metabolism may also exist in LSC vs. non-LSC. To determine if sphingolipids play a functional role in AML biology, we transplanted mice with peripheral blood cells from 13 AML patients, including those with therapy resistant and relapsed disease, and treated engrafted mice with myriocin or FTY720. We observed heterogeneous responses in our cohort, with reduction of leukemic burden in 3 and 5 samples following treatment with FTY720 and myriocin, respectively. Remarkably, serial transplantation of FTY720 responders into untreated secondary mice at limiting dilution demonstrated decreased LSC frequency in FTY720-treated primary mice compared to vehicle-treated controls, whereas myriocin responders showed no alteration of LSC frequency. These results suggest that FTY720 but not myriocin treatment affects LSC number and/or function. To stratify responders from nonresponders, we performed transcriptional analysis of untreated patient samples and compared these data to transcriptional signatures generated from the normal and AML hierarchy. Bioinformatic analysis demonstrated that FTY720 responders had an enriched LSC signature compared to nonresponders. In contrast, myriocin responders exhibited a strong GMP signature compared to nonresponders. Thus, normal human hematopoietic stem and progenitor cells display a variable dependence on sphingolipid biology that is also distinct between LSC and HSC, pointing to targeting of bioactive sphingolipids as a novel therapeutic strategy in AML to eradicate LSC while sparing HSC. Citation Format: Stephanie Z. Xie, Elisa Laurenti, Robin Ferrari, John E. Dick. Differential dependence on sphingolipid metabolism in the normal and leukemic human hematopoietic hierarchy. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A04.

Targeting MTHFD2 in Acute Myeloid Leukemia

Alterations in differentiation pathways contribute to the development of acute myeloid leukemia (AML). Differentiation therapy with all-trans retinoic acid (ATRA) has dramatically altered the treatment of acute promyelocytic leukemia, transforming it from a nearly fatal disease to a curable one. We set out to identify cellular pathways that contribute to AML differentiation, with the goal of identifying new therapeutic targets. We analyzed gene expression data from AML cell lines treated with phorbol 12-myristate 13-acetate (PMA), ATRA, Vitamin D, the BET inhibitor JQ1 and the DOT1L inhibitor EPZ00477, treatments known to induce AML differentiation and impair growth. Folate-mediated one-carbon metabolism was one of only three metabolic pathways altered by these compounds, with expression of MTHFD2 consistently downregulated with each compound.MTHFD2 is an NAD-dependent, bi-functional mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase. It is differentially expressed in embryonic and transformed tissues and is upregulated in myeloid progenitors. MTHFD2 is the most differentially expressed metabolic enzyme in cancer cells versus normal cells, including normal proliferating cells. We thus investigated the role of MTHFD2 in myeloid malignancy. First, we demonstrated using ChIP-qPCR, MYC knockdown and MYC inhibition with a BET inhibitor, that MYC directly regulates MTHFD2 expression in AML. Knockdown of MTHFD2 with two shRNAs confirmed to have on-target activity, induced myeloid differentiation in AML cell lines, as measured by Cd11b expression, morphologic changes and induction of a previously validated AML differentiation gene expression signature. MTHFD2 knockdown decreased cell growth in AML cell lines, as well as decreased colony formation in methylcellulose in both AML cell lines and primary patient blasts. AML cells transduced with these two MTHFD2-directed shRNAs demonstrated attenuated growth in an orthotopic mouse model of AML. Furthermore, three MTHFD2-directed shRNAs prolonged survival in an MLL-AF9 mouse leukemia model. Additionally, using a doxycycline inducible shRNA system, we demonstrated that two miR30-shRNAs directed against MTHFD2 decreased AML burden in mice with established disease and prolonged survival.To identify biomarkers of response to MTHFD2 suppression, we used single sample Gene Set Enrichment Analysis (ssGSEA) to identify primary patient AML samples enriched for gene expression signatures of folate-mediated one-carbon metabolism and MTHFD2. We found in both independent data sets that the cluster of patients enriched for expression of the one-carbon folate pathway gene signatures was also enriched for patients with FLT3-ITD mutations, a subset of AML with a particularly poor prognosis. In addition, in an shRNA screen targeting 11,194 genes and performed in 216 cancer cell lines, including 17 AML lines, FLT3-ITD was a biomarker of response to MTHFD2 knockdown. We next validated that while MTHFD2 suppression induced measureable differentiation in all six AML cell lines examined, it induced the most robust induction of apoptosis in FLT3-ITD mutant AML. The mitochondrial one-carbon folate pathway is thought to contribute to cellular oxidative balance by providing reducing power in the form of NAD(P)H, and suppression of MTHFD2 was previously shown to increase ROS levels. Indeed, suppression of MTHFD2 led to a marked increase in ROS in the FLT3-ITD positive AML cell lines in which apoptosis was induced.In summary, a decrement in MTHFD2 expression was found at the center of multiple AML perturbations that impair AML growth and induce differentiation. Our data support MTHFD2 as an AML dependency and FLT3-ITD as a potential biomarker of response. We thus provide critical preclinical evidence for targeting of MTHFD2 as a therapeutic strategy in AML. DisclosuresStone:Celgene: Consultancy; Merck: Consultancy. DeAngelo:Celgene: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy. Stegmaier:Novartis Pharmaceuticals: Consultancy.

P38MAPK Inhibition Blocks Inflammatory Signaling in Acute Myeloid Leukemia

Background: p38 mitogen-activated protein kinase (p38MAPK) is activated by various pro-inflammatory and stress-related stimuli, and has been an attractive therapeutic target for autoimmune diseases. p38MAPK (hereafter referred to as p38) signaling is also involved in cell proliferation, differentiation, apoptosis, and invasion, suggesting that it may be a potential therapeutic target for cancer. We found that inflammatory cytokines, including interleukin-1 (IL-1), promote growth and survival of more than half of the acute myeloid leukemia (AML) patient samples we tested. Since p38 is a downstream mediator of inflammatory pathways, we hypothesized that targeting p38 might be an effective therapeutic strategy in AML and other hematologic malignancies. To test this hypothesis, we evaluated the effectiveness of three p38 inhibitors using in vitro studies in primary AML patient samples. We found that targeting p38 blocks IL-1-activated extrinsic signaling and is a critical therapeutic target in a large subset of AML patients.Methods: We screened ~1000 primary leukemia patient samples for sensitivity to p38 inhibition using varying concentrations of doramapimod (BIRB 796) in a cell growth assay. We compared the sensitivity profile of doramapimod with 2 other small-molecule p38 inhibitors currently in clinical trials: ARRY 614, a dual p38/Tie2 inhibitor, and ralimetinib, which blocks activation of p38 by its substrate MK2. We determined cell viability, survival, and downstream signaling in the presence of 10 ng/ml IL-1α or IL-1β. Patient samples with IC50 < 1000nM were considered drug responsive.Results: In our patient population, we observed response rates of 31% in AML (109/350), 27% in myelodysplastic syndromes (MDS; 25/93), 19% in myeloproliferative neoplasms (23/123), 13% in mature B-cell neoplasms (30/232), and 10% in precursor lymphoid neoplasms (19/182). Focusing on AML, we compared the sensitivity profile of doramapimod with two other small-molecule p38 inhibitors, ralimetinib and ARRY 614. These inhibitors showed strikingly similar sensitivity profiles to doramapimod when tested in an additional 25 primary AML samples, with ~25% responsive and median IC50 of 11 nM for ARRY 614 (range: 7-650nM), 105 nM for ralimetinib (range: 7-850nM), and 18 nM for BIRB 796 (range: 13-40nM). Because IL-1 is known to stimulate p38 signaling, we compared the response rates for these three p38 inhibitors with or without IL-1 in a dose-response study. IL-1 increased the percent of AML samples responding to p38 inhibition from 25% to 60%, indicating a potentially important role of extrinsic inflammatory stimuli in p38 inhibitor sensitivity. Consistent with this all three p38 inhibitors were similarly effective in blocking the growth of primary AML CD34+ progenitors, suggesting that targeting p38 might reduce early progenitor AML cells. Further, we compared doramapimod, ralimetinib, and ARRY 614 for their ability to inhibit p38 phosphorylation in primary AML samples using flow cytometry and immunoblot analysis; all three inhibitors blocked p38 pathway activation in AML cells. Notably, in clinical studies of ARRY 614 in MDS patients, preliminary biomarker analyses demonstrated persistent inhibition of phospho-p38 in the bone marrow during the treatment. Also, consistent with functional inhibition of p38, there was a profound decrease in plasma cytokine concentrations, most significantly IL-1, during ARRY 614 treatment. In 250 primary AML samples, we observed no correlation between BIRB 796 sensitivity in vitro and clinical metrics such as white blood cell count, blast percentage in peripheral blood or bone marrow, karyotype, or tumor genotype. This suggests that IL-1 and p38 activation might be independent biomarkers of drug sensitivity.Conclusions: These data underscore the importance of the p38MAPK pathway in the pathobiology of AML and provide strong preclinical evidence to support p38MAPK as a therapeutic target. Targeting p38MAPK might also block tumor-extrinsic signaling, as indicated by IL-1-activated signaling. That all three p38MAPK inhibitors showed comparable sensitivity profiles holds promise for ARRY614, which showed the lowest median IC50 and is currently in clinical development. In addition, with further study these findings may be extended to hematologic malignancies other than AML. DisclosuresWinski:Array BioPharma Inc.: Employment. Cable:Array BioPharma Inc.: Employment. Tyner:Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding. Agarwal:CTI BioPharma: Research Funding.

Novel Leukemia Stem Cell-Targeted Therapy for Acute Myeloid Leukemia Based on Dual Inhibition of Ezh1/Ezh2

Acute myeloid leukemia (AML) is a clonal malignant disorder originating from a small number of leukemic stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of LSCs is a promising strategy for the prevention and treatment of AML relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the stemeness of ES cells and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) is a catalytic component of PRC2 that trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Mutations and overexpression of EZH1/2 are associated with cancers including hematopoietic malignancies. Here, we used genetic deletion of EZH1/2 or a novel dual inhibitor of EZH1/2 activity to show that loss or inhibition of EZH1/2 eradicates dormant AML stem cells.To examine the effects of genetic deletion of EZH1/2 on AML cells, Ezh1-null, Ezh2-conditional and double knock-out mice were generated. Hematopoietic stem/progenitor cells prepared from single knock-out mice or double knock-out mice were transduced with various types of AML fusion-genes, such as MOZ-TIF2, MLL fusions, AML1-ETO, and others by retroviral infection, and cultured in vitro or transplanted into irradiated recipient mice to induce AML in vivo. When the cells were cultured in vitro, double deletion of Ezh1/2 induced cell differentiation and apoptosis more severely than single deletions in all subtypes of AML tested, resulting in complete loss of cells. In AML mice, deletion of Ezh1/2 induced AML cell differentiation and complete remission of AML, which was not achieved by single deletion of Ezh1 or Ezh2. Genetic deletion of both Ezh1 and Ezh2 on the LSC fraction dramatically reduced the number of LSCs (Lin-c-Kit+ CD16/32+ CD34+, L-GMP), especially quiescent LSCs, whereas deletion of either Ezh1 or Ezh2 did not have such a strong effect. The transcriptional profiles of LSCs deficient in both Ezh1 and Ezh2 were characterized by the upregulation of cell cycle-related genes such as Cyclin D1/D2, which is the main regulator of G0/G1 transition, along with differentiation-related genes. These results suggested that deletion of both Ezh1 and Ezh2 is required for eradication of LSCs.To investigate whether pharmacologic inhibition of EZH1/2 could serve as a therapeutic strategy in AML, we developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1 and EZH2. The drug induced cell differentiation and apoptosis in most subtypes of AML tested in vitro and its effects were similar to those of genetic depletion of EZH1/2. A selective EZH2 inhibitor did not affect the growth and survival of AML cells to the same extent as the dual inhibitor. Oral administration of the EZH1/2 dual inhibitor reduced the number of LSCs effectively in AML mice in a manner similar to the effect of genetic deletion of EZH1/2.Taken together, these results strongly suggest that dual inhibition of EZH1 and EZH2 is a promising therapeutic strategy to eradicate LSCs in a wide range of AMLs, which could lead to important advances in the treatment of AML. DisclosuresHonma:Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: Implications for leukemia therapy.

Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI-1 in AML and the effects of a novel small molecule selective inhibitor of BMI-1, PTC-209. BMI-1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse-phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI-1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI-1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC-209 reduced protein level of BMI-1 and its downstream target mono-ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. PTC-209 induced apoptosis in patient-derived CD34(+)CD38(low/-) AML cells and, less prominently, in CD34(-) differentiated AML cells. BMI-1 reduction by PTC-209 directly correlated with apoptosis induction in CD34(+) primary AML cells (r = 0.71, P = 0.022). However, basal BMI-1 expression was not a determinant of AML sensitivity. BMI-1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition

AbstractCancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34+ progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GACK320A allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

Abstract 3039: Targeting mitochondrial DNA polymerase gamma (POLG) as a novel therapeutic strategy for acute myeloid leukemia (AML)

Abstract Mitochondria contain their own 16.6kb genome (mtDNA) which encodes 13 protein-coding genes essential for electron transport chain activity. In mammalian cells, it is replicated solely by the nuclear-encoded mitochondrial DNA polymerase gamma (POLG), which exists as a heterotrimer with POLG2 to perform replication and repair of mtDNA. Through bioinformatic analyses, we observed increased POLG expression in a subset of AML cell lines compared to other cancer cell lines. As a subset of AML patients also have unique mitochondrial characteristics, including increased mitochondrial mass and mtDNA content compared to normal hematopoietic stem cells (Skrtic et. al., Cancer Cell, 20:674, 2011), we addressed the effects of targeting POLG as a novel therapeutic strategy for AML. OCI-AML2 and TEX leukemia cells were treated with the anti-retroviral drug 2′,3′-dideoxycytidine(ddC) that exhibits off-target inhibition of POLG catalytic activity. At concentrations as low as 200nM, ddC depleted mtDNA, decreased mRNA expression of mtDNA transcripts, reduced the expression of mitochondrial encoded proteins COX 1 and 2 subunits that form the catalytic core of respiratory chain complex IV, decreased basal oxygen consumption rate (OCR), and reduced the proliferation and viability of AML cells. However, AML cells had large reserves in their mtDNA; as significant changes in mitochondrial proteins, metabolism, proliferation and viability were only observed with a threshold of &amp;gt;95% depletion of mtDNA. In contrast, lesser reductions in mtDNA content with ddC did not significantly affect levels of mitochondrial transcripts, metabolism, or cell viability. Next, we used a genetic approach and explored the impact of knocking down POLG with multiple independent shRNA in AML cells. POLG knockdown in OCI-AML2 cells depleted mtDNA content by 60% compared to controls. Consistent with the findings with ddC, mtDNA depletion to this extent produced only quantitatively minor changes in the protein expression of the mtDNA-encoded COX 1 and 2 and basal OCR. Despite the minimal effect on mitochondrial metabolism, POLG knockdown decreased the growth and viability of OCI-AML2 cells, and increased apoptosis, as measured by Annexin V staining. Results of electron microscopy analyses indicate that POLG knockdown disrupted mitochondrial cristae structure compared to controls. In summary, these results indicate that POLG plays a role in maintaining AML cell viability and mitochondrial structure and this function is independent of mtDNA replication and oxidative metabolism. Citation Format: Sanduni Liyanage, Rose Hurren, Rebecca Laposa, Aaron Schimmer. Targeting mitochondrial DNA polymerase gamma (POLG) as a novel therapeutic strategy for acute myeloid leukemia (AML). [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 3039. doi:10.1158/1538-7445.AM2015-3039

Regulation of the C/EBPα signaling pathway in acute myeloid leukemia (Review).

The transcription factor CCAAT/enhancer binding protein α (C/EBPα), as a critical regulator of myeloid development, directs granulocyte and monocyte differentiation. Various mechanisms have been identified to explain how C/EBPα functions in patients with acute myeloid leukemia (AML). C/EBPα expression is suppressed as a result of common leukemia-associated genetic and epigenetic alterations such as AML1-ETO, RARα-PLZF or gene promoter methylation. Recent data have shown that ubiquitination modification also contributes to its downregulation. In addition, 10-15% of patients with AML in an intermediate cytogenetic risk subgroup were characterized by mutations of the C/EBPα gene. As a transcription factor, C/EBPα can translocate into the nucleus and further regulate a variety of genes directly or indirectly, which are all key factors for cell differentiation. This review summarizes recent reports concerning the dysregulation of C/EBPα expression at various levels in human AML. The currently available data are persuasive evidence suggesting that impaired abnormal C/EBPα expression contributes to the development of AML, and restoration of C/EBPα expression as well as its function represents a promising target for novel therapeutic strategies in AML.

Mass Cytometric Analysis of AML Stem and Early Progenitor Cells Reveals Karyotype and Genotype-Specific Immunophenotypes That May Represent Targets for Antibody-Directed Therapies

Introduction: Leukemic stem cells (LSCs) are recognized as important mediators of leukemia relapse. Thus, therapeutic antibodies are in development to target antigens present on these cells. The success of this approach relies on a detailed understanding of the surface marker expression patterns of LSCs. To address this, we applied multi-parametric single-cell mass cytometry (MCM) to deeply profile the surface marker expression of all major immunophenotypic populations in bone marrow aspirates (BM) from patients with acute myeloid leukemia (AML).Methods: BMs from 41 leukemia patients (30 AML, 4 APL, 2 high-risk MDS, 5 AML in CR) and 5 healthy donors and were processed immediately after aspiration (<1min) and stored for pooled analysis with two overlapping 39-antibody MCM panels (50 markers total). All samples were barcoded, such that 20 samples (leukemia and healthy) could be combined into a single tube for simultaneous antibody staining and analysis, resulting in high precision (coefficient of variation = 10-20%).Results: Distinct AML subtype-specific patterns of cell frequencies across immunophenotypic populations were detected. Patients with core binding factor (CBF) AML (n=5) and those with adverse-risk karyotpic abnormalities (n=6) exhibited the greatest expansion of immunophenotypic hematopoietic stem cells (HSCs) and early progenitors (MPP, CMP), while patients with APL (n=4) and FLT3-ITD normal karyotype AML (NK-AML; n=11) exhibited reduced expansion of early progenitors and expansion in more mature myeloid progenitors (GMP, myelo-monoblasts).As a result of barcoding, high resolution (2-3 fold changes) measurement of surface marker expression detected multiple aberrancies across almost all identifiable immunophenotypic populations. Specifically, several genotype- and karyotype-specific trends in aberrant marker expression were observed in hematopoietic stem and progenitor cell populations (HSPCs; CD34+CD38low). FLT3-ITD samples were characterized by increased CD7, CD33, CD123, CD45, CD321, and CD99, as well as decreased CD34, CD117, and CD38. FLT3wt NK-AML samples (n=6) were characterized by increased CD99 as well as decreased CD71, CD47, CD34, and CD45. Adverse-risk karyotype samples (n=6) were characterized by increased CD99 and decreased CD47. All p values were significant (ranging from 0.02 to 4.5x10-7).While all 36 samples (AML or APL) displayed immunophenotypic abnormalities within the CD34+CD38low gated population (3.6 abnormalities on average), there were 9 samples in which an unambiguous separation into normal and leukemic populations was observed among the cells in this gate. Interestingly, among these samples, some markers were aberrantly high in leukemic cells of one subtype, and aberrantly low in leukemic cells of another, e.g. HLA-DR was extremely high in HSPCs of sample #22 (MLL rearrangement), and aberrantly low in all 4 APL samples. CD99 was most consistently elevated in the AML samples (28/36), but was normal in 6 samples, including a majority of those with t(8;21).These results suggest that therapeutic antibodies directed against molecules such as CD33 may be less effective for AML subtypes such as FLT3wt NK-AML (~40% decrease in CD33), and more effective for other subtypes such as FLT3-ITD NK-AML (7-fold increase in CD33). HSPCs from patients with FLT3-ITD mutations also displayed a 20-fold increase in CD123, which could also be therapeutically targeted. These findings, combined with our recent observation that HSPCs from FLT3-ITD NK-AML patients have an extremely low S-phase fraction, may provide a mechanistic basis for the improved disease-free survival recently reported for FLT3-ITD NK-AML patients treated with fractionated gemtuzumab ozogamicin in combination with standard therapy.Conclusions: We have developed an innovative MCM approach for the analysis of hematologic malignancies and demonstrated that different genotypic and karyotypic subsets of AML are highly varied in their immunophenotypic properties, particularly within the stem and progenitor cell compartment. These data also suggest that antigens that distinguish AML stem cells from normal stems cells are likely to be karyotype and genotype-specific. These findings have important implications for the design of therapeutic strategies in AML. [Display omitted] [Display omitted] [Display omitted] DisclosuresBehbehani:Fluidigm: Consultancy. Medeiros:Agios: Consulting - Ad board Other. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

The Role of pp90rsk-Mediated CREB Phosphorylation in Acute Myelogenous Leukemia

CREB (cAMP Response Element Binding Protein) is a nuclear transcription factor that plays a critical role in the pathogenesis of Acute Myeloid Leukemia (AML). CREB is overexpressed in the majority of AML patients, and this is associated with a poor prognosis. CREB overexpression leads to increased AML cell proliferation and resistance to apoptosis in vitro. For CREB to be transcriptionally active, however, it must first be phosphorylated at Serine 133. Previous work has suggested that Ribosomal S6 Kinase (pp90rsk or RSK) is the primary kinase responsible for growth factor-induced phosphorylation of CREB, and that RSK is activated downstream of the Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-Colony Stimulating Factor (G-CSF) in AML cells. The overall role and regulation of RSK in AML cells, however, remains unknown. Thus, the goal of this study was to characterize the RSK-CREB signaling pathway in AML, with the overall hypothesis that disruption of this pathway represents a potential therapeutic strategy for the treatment of AML. We report that of the four known isoforms of RSK, RSK1 and RSK2 appear to be the predominant subtypes expressed in AML cells. To identify additional upstream pathways responsible for activation of these isoforms in AML cells, we performed cytokine stimulation experiments. Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Thrombopoietin (TPO), and Interleukin 3 (IL-3) were all capable of stimulating phosphorylation and activation of RSK in KG-1 and HL-60 cells, implicating that multiple signaling pathways converge on RSK. G-CSF and GM-CSF had the greatest effects, stimulating increased phosphorylation and activation of RSK by 2.8 and 2.6 fold, respectively. Using shRNA technology, we then generated AML cell lines (HL-60 and KG-1) in which the expression of each isoform was 'knocked-down’ to examine whether these two isoforms play unique roles in AML cells. Interestingly, RSK1 appears to be the isoform primarily responsible for phosphorylating CREB downstream of the G-CSF receptor. We demonstrate that G-CSF treatment of RSK1 knockdown cells did not induce an increase in CREB phosphorylation, and baseline CREB phosphorylation was also significantly decreased in these cells. Previous data had shown that blockade of total RSK activity using the non-selective RSK inhibitor BI-D1870 induced cell death in both AML cell lines and primary AML patient samples. RSK1 knockdown in HL-60 cells sensitized them to this agent (IC50 1.2 microM vs 3.3 microM), while the sensitivity of RSK2 knockdown cells was unchanged. Finally, since targets of RSK also include regulators of apoptosis (BAD) and cellular stress signaling pathways (IkB), we examined the effects of inhibiting RSK on the phosphorylation of these proteins. Levels of phosphorylated CREB and BAD decreased by 50% in HL-60 cells after 2 hours of treatment with the RSK inhibitor, suggesting that this treatment induces apoptosis. In summary, targeting the RSK-CREB signaling axis may represent a novel therapeutic strategy for AML. Future experiments will further define the role of RSK in proliferation and survival of AML cells and normal hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Co-Activation of AMPK and mTORC1 Is Synthetically Lethal in Acute Myeloid Leukemia

Virtually all recurrent molecular alterations in acute myeloid leukemia (AML) functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as patients, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML.Our studies employed a novel AMPK activator called GSK621. Using CRISPR and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, as demonstrated by western blotting, immunofluorescence, flow cytometry and electron microscopy. Blocking autophagy via shRNA-mediated knockdown of ATG5 and ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays.Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation in AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1 both directly (through raptor phosphorylation) and indirectly (through TSC2 phosphorylation), sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. This protective effect was mediated by mTORC1-dependent modulation of the ATF4/CHOP stress response pathway. The ultimate functional consequence was that, rather than diminishing GSK621-induced cytotoxicity, persistent mTORC1 activity was in fact synthetically lethal with AMPK activity in AML cells. This synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. It could also be enhanced in AML cells by mTORC1 overactivation induced by CRISPR-mediated deletion of TSC2.Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. This finding may have important implications for AMPK and mTORC1 signaling pathways in cancer biology more broadly. Context-dependent permissiveness towards mTORC1 activation may amplify the response to cytotoxic stress, such as that resulting from AMPK activation by GSK621. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials. DisclosuresBrusq:GSK: Employment. Nicodeme:GSK: Employment.