Murine Acute Myeloid Leukemia Cells Research Articles

Tight Regulation of H3K79 Methylation Levels in KMT2A-Rearranged AML

Rationale: KMT2A-rearrangements (KMT2A-r) in acute myeloid leukemia (AML), encompassing both KMT2a-fusions (KMT2A-F) and KMT2A-partial tandem duplications (KMT2A-PTD), represent a subgroup of AML with a particularly poor prognosis. Both KMT2A-F and KMT2A-PTD share a dependency on the H3K79 methyltransferase DOT1L for proper histone 3 lysine 79 dimethylation (H3K79me2) on target genes such as HOXA9 and MEIS1. Pharmacologic inhibition of DOT1L results in downregulation of KMT2A fusion/PTD target genes. Albeit rare, complete responses observed in patients with relapsed/refractory KMT2A-r leukemia treated with a DOT1L inhibitor underscore the clinical relevance of this pathway. The second most commonly co-occurring mutation with KTM2A-PTD are mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2). The canonical oncogenic function of mIDH1/2 involves aberrant production of the oncometabolite 2HG and inhibition of TET2. However, 2HG also induces histone hypermethylation, including H3K79 hypermethylation as reported by several groups. Based on this, we sought to study the effect of increasing H3K79 methylation in KMT2A-r AML either via direct overexpression of DOT1L or introduction of mIDH1/2.Results: As increased H3K79 methylation has only been observed upon 2HG exposure or mIDH1/2 expression in experimental systems, we analyzed H3K79 methylation in primary AML patient samples. We found increased H3K79 methylation in IDH1/2 mutant AML at diagnosis. Given the high percentage of IDH1/2 mutations in KMT2A-PTD leukemias, we sought to understand what effect increasing H3K79me2 would have on KMT2A-r leukemia. To this end, we overexpressed DOT1L in KMT2A-MLLT3 murine AML cells. Surprisingly, we found H3K79me2 was counterselected, and DOT1L overexpressing cells grew slower compared to controls. Furthermore, ChIP-Seq revealed that DOT1L overexpression increased H3K79me2 peak height and width outside KMT2A-fusion target genes, effectively blunting the difference between fusion targets and other genomic loci. This was associated with a decreased expression of target genes. This counterintuitive finding led us to analyze global (rather than locus specific) H3K79 methylation in AML patient samples. We found that H3K79me2 is globally low in KMT2A-r AML. This constellation is reminiscent of CALM-AF10 AML, which typically loses the second AF10 allele, resulting in globally low H3K79me2/3, preserved/increased methylation on fusion target loci, and DOT1L dependence. We next tested the effect of IDH mutations on KMT2A-r AML. Co-expression of mIDH1 and MLL-MLLT3 in murine lin-sca1+ckit+ bone marrow cells resulted in an aggressive AML that was dependent on DOT1L, but not mIDH1. As we found when DOT1L was overexpressed, H3K79me2 was counterselected in the mouse. This finding was confirmed by introducing KMT2A-MLLT3 into mIDH2 knock-in bone marrow recapitulating the order of mutations in AML patients, as well as by overexpressing mIDH1 in a KMT2A-PTD cell line. In two patient samples with KMT2A-PTDs and IDH1/2 mutations, we found no difference in H3K79 methylation compared to non-IDH1/2 mutant KMT2A-PTD samples.Summary: While two groups have reported cooperation of IDH1/2 mutations with retroviral overexpression of KMT2A targets (HOXA9/Meis1), we find that there is no cooperation between KMT2A-rearrangements themselves and mIDH1/2. This may in part be due to H3K79 hypermethylation, which is not tolerated in KMT2A-r disease, while HOXA9/Meis1 retroviral models are insensitive to H3K79 modulation. Interestingly, H3K79me2 is globally low in KMT2A-F patient samples. Our findings suggest that KMT2A-F leukemias maintain tight control over H3K79me2, and neither increases nor decreases are tolerated. This may have implications for the clinical/biomarker development of DOT1L directed therapies. DisclosuresBernt:GSK: Other: husband works at GSK.

MiR-193a Is a Negative Regulator of Hematopoietic Stem Cells and Promotes Anti-Leukemic Effects in Acute Myeloid Leukemia

The microRNA (miRNA) family miR-193 consists of two members, miR-193a and miR-193b, which share identical seed regions and are therefore thought to be functionally redundant. However, the target spectrum of a miRNA is defined by the mRNA spectrum of the tissue, where it is expressed as well as dynamic strand preferences (miRNA-5p vs miRNA-3p) (Kuchenbauer et al., Blood, 2011). We and others previously showed that miR-193b is a tumor suppressor in acute myeloid leukemia (AML) (Bhayadia et al., JCO, 2018) and a potent regulator of hematopoietic stem cell expansion (Haetscher et al., Nat Com, 2015). However, the role of miR-193a in normal and malignant hematopoiesis is still unclear.First, we profiled the expression of miR-193a-5p and miR-193a-3p in human hematopoietic subpopulations from healthy donors via quantitative real-time PCR (qRT-PCR). Unlike miR-193b, which is enriched in hematopoietic stem cells (HSC) (Haetscher et al., Nat Com, 2015), miR-193a expression was restricted to differentiated cells of the myeloid lineage (granulocytes, monocytes, and erythrocytes). To investigate the function of miR-193a during normal hematopoiesis, we analyzed its effect on a murine myeloid progenitor cell line (32D cells) upon engineered overexpression of miR-193a in combination with granulocyte-stimulating growth factor (G-CSF) treatment (n=3-7). MiR-193a strongly promoted granulocytic differentiation of 32D cells already after two days compared to the control arm (p=0.006) as assessed by flow cytometry and morphological analysis. To analyze the effect of miR-193a on HSC function, we ectopically expressed miR-193a in highly purified E-SLAM (CD45+EPCR+CD48-CD150+) cells and transplanted them into lethally irradiated recipient mice (n=5 mice/arm). HSCs overexpressing miR-193a failed to reconstitute hematopoiesis (p=0.038). The negative effect on stem cell properties could be translated to human CD34+ cord blood (CB) cells, where miR-193a overexpression significantly reduced colony counts. Taken together, these results suggest, that miR-193a has pro-differentiation and anti-stemness functions.To assess the role of miR-193a in AML, we profiled miR-193a-5p and miR-193a-3p in two cohorts of de novo pediatric (n=187) and de novo adult AML patients (n=40) by qRT-PCR. We found that both miRNA strands (miR-193a-5p and miR-193a-3p) are present at similar levels, which is in contrast to the almost undetectable levels of miR-193b-5p strand, hinting at additional functional roles for miR-193a through its 5p arm. MiR-193a was significantly downregulated in adult and pediatric AML compared to healthy donor samples, suggesting a possible tumor suppressor function. To investigate the role of miR-193a in AML, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9/Meis1 (aggressive, short latency in vivo) with low endogenous miR-193a levels. Restoration of miR-193a by lentiviral overexpression delayed Hoxa9/Meis1 mediated leukemogenesis in vivo (p=0.01, n=8-10). Furthermore, miR-193a overexpression reduced leukemic growth of human AML cell lines (n=6) and decreased colony-forming capacity of primary AML patient samples (n=4; p=0.029) in vitro. To identify novel targets of miR-193a, we performed a proteomics screen in human NOMO1 AML cells overexpressing miR-193a compared to an empty control vector (n=5). We identified and verified stathmin (STMN1), a tubulin-associated, intracellular phosphoprotein previously linked to proliferation of AML cells, as a novel putative miR-193a target, further explaining its tumor suppressor effect.Taken together, we characterized miR-193a as a positive regulator of myeloid differentiation and negative modulator of HSCs. Based on the balanced presence of both miRNA arms, we hypothesize that each arm is functionally active and has different functions such as pro-differentiation and anti-stemness. Furthermore, this work is the first characterization of a miRNA family that exerts cooperative effects at both early and late hematopoietic differentiation stages, highlighting a novel mechanism of balancing anti-stemness and pro-differentiation. DisclosuresDöhner:Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau.

Abstract 2455: The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC)

Abstract Introduction: Prognosis for AML patients (pts) is inadequate. Therapeutic strategies are needed to target not only bulk blasts but also the distinct LSCs, which are more resistant to chemotherapy & cause relapse. lncRNAs are non-coding RNA transcripts longer than 200 nucleotides that regulate cellular processes. A functional role in LSCs has yet to be elucidated. Methods: We derived a LSC-specific lncRNA profile using whole transcriptome sequencing (RNA-seq) data of 377 pts (<60 years) with cytogenetically normal (CN) AML. These data were correlated with a 'core enriched' (CE) gene expression signature representing 44 genes deregulated in LSCs (Eppert, Nat.Med. 2011). DANCR expression in the LSC-enriched compartment was validated using qRT-PCR. Transferrin/antibody conjugated nanoparticle (NP) knock down (KD) was used as an efficient & less toxic avenue to deliver siRNA against DANCR or scramble (SCR) in vitro & in vivo. LSC features (self-renewal, quiescence, engraftment) were analyzed & frequency determined in 3 primary AML samples and a CN-AML (Flt3ITD/WT/MllPTD/WT) mouse model. Results: 161 lncRNAs were consistently deregulated in CEhigh pts with a p<.001 & a correlation coefficient >.5 (Spearmen correlation test). We chose DANCR to further analyze because it was among the top lncRNAs upregulated in CEhigh pts, is highly conserved between human & mouse, & was previously shown to have a role in promoting stemness in hepatocellular carcinoma (Yuan, Hepatology 2016). We confirmed that DANCR expression was significantly higher in functionally validated LSC enriched populations (p=.05). To assess the role in self-renewal, we KD DANCR in pts cells & found a decrease in colony numbers after replating (average decrease vs SCR: 38.9%, p=.03). Using membrane labeling assays, we found a decrease of quiescent cells after DANCR KD (average decrease: 36.1%, p=.04). Long term colony-initiating cell cultures were performed to determine the LSC frequency & we found significant decreases in the number of LSCs in primary pts samples after DANCR KD (average decrease of LSC frequency: 79.2%, p=.001). Mice transplanted with murine AML cells (CD45.2+) were treated with NP (n=3 per group) for 5 days (d). 2d later, bone marrow was harvested & retransplantated (Tx) using limiting dilutions (2x106, 1x106, 5x105) into irradiated BoyJ (CD45.1+) recipients (n=30). 14d post-Tx, anti-Dancr recipient mice showed a lower engraftment (CD45.2/CD45.1: 37% vs 47%, p=.03) and also a concomitant improvement in survival (p<.001). Conclusion: We show for the first time that LSCs have a distinct lncRNA profile. Among the highly expressed lncRNAs associated with LSCs we identified & validated DANCR. Our data show that DANCR has an impact on LSC function (self-renewal, quiescence, engraftment) & regulates LSC frequency. DANCR is the first lncRNA shown to have a functional role in LSCs & represents a novel target for AML treatment. Citation Format: Marius Bill, Malith Karunasiri, Jessica Kohlschmidt, Allison E. Walker, Zachary Brannan, Stefano Volinia, Clara D. Bloomfield, Ramiro Garzon, Adrienne M. Dorrance. The long non-coding RNA (lncRNA) DANCR in acute myeloid leukemia (AML) stem cells (LSC) [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 2455.

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

ALOX5 exhibits anti-tumor and drug-sensitizing effects in MLL-rearranged leukemia

MLL-rearranged acute myeloid leukemia (AML) remains a fatal disease with a high rate of relapse and therapeutic failure due to chemotherapy resistance. In analysis of our Affymetrix microarray profiling and chromatin immunoprecipitation (ChIP) assays, we found that ALOX5 is especially down-regulated in MLL-rearranged AML, via transcription repression mediated by Polycomb repressive complex 2 (PRC2). Colony forming/replating and bone marrow transplantation (BMT) assays showed that Alox5 exhibited a moderate anti-tumor effect both in vitro and in vivo. Strikingly, leukemic cells with Alox5 overexpression showed a significantly higher sensitivity to the standard chemotherapeutic agents, i.e., doxorubicin (DOX) and cytarabine (Ara-C). The drug-sensitizing role of Alox5 was further confirmed in human and murine MLL-rearranged AML cell models in vitro, as well as in the in vivo MLL-rearranged AML BMT model coupled with treatment of “5 + 3” (i.e. DOX plus Ara-C) regimen. Stat and K-Ras signaling pathways were negatively correlated with Alox5 overexpression in MLL-AF9-leukemic blast cells; inhibition of the above signaling pathways mimicked the drug-sensitizing effect of ALOX5 in AML cells. Collectively, our work shows that ALOX5 plays a moderate anti-tumor role and functions as a drug sensitizer, with a therapeutic potential, in MLL-rearranged AML.

Targeting Innate and Adaptive Immune Responses for the Treatment of Acute Myeloid Leukemia

Background: Successful immunotherapeutic approaches for acute myeloid leukemia (AML) have yet to be developed. We hypothesized that targeting both the innate and adaptive immune responses in leukemic hosts would elicit significant anti-tumor activity with lesser toxicities than chemotherapy. To test this, we evaluated the efficacy of immune checkpoint inhibition (murine anti-PD-1 antibody (ab)) alone and in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an innate immune agonist and anti-vascular agent, in an immunocompetent model of murine AML.Methods: Expression of PD-L1 was assessed by flow cytometry on the murine AML cell line, C1498, alone and following treatment with vehicle, DMXAA or interferon-gamma (positive control). A LEGEND MAX mouse ELISA kit was utilized to measure IL-6 and IFN-β. C57BL/6 mice were inoculated with stably transfected C1498 murine AML cells expressing luciferase and the fluorescent protein DSRed2. Once disease was established, animals were treated with vehicle, DMXAA (20 mg/kg every four days x 7 weeks), anti-murine PD-1 antibody (10 mg/kg every 3 days x 4 doses) or DMXAA + anti-PD-1 antibody (same doses). Animals underwent weekly clinical assessments, weights, and bioluminescent imaging for disease burden. Overall study endpoints were time to morbidity and differences in leukemia disease burden as compared with vehicle-treated controls. Mice were euthanized on day 15 after injection of C1498 cells (8 days following treatment) for collection of plasma, bone marrow, liver and spleen samples for tumor burden, activated T-cells.Results: DMXAA doses (ranging from 1-100 μg/ml) inhibited C1498 in vitro cell growth at 48 hours (48h) in a dose dependent manner. Treatment of C1498 cells in culture with escalating doses of DMXAA (1-100μg/ml) or IFN-gamma (positive control) induced higher PD-L1 expression on these AML cells consistent with direct immunomodulatory effects. Furthermore, C1498 cells exposed to higher doses of DMXAA (10-100μg/ml) for 48h produced measurably higher levels of IL-6 and IFN-β expression in cell supernatants.We then examined the effects of DMXAA, anti-PD-1 ab, or the combination of DMXAA + anti-PD-1 ab treatment in vivo in C57BL/6 mice systemically engrafted with C1498-luciferase AML cells. Treatment overall was well tolerated and resulted in significantly decreased disease burden as measured by total body bioluminescence vs. vehicle controls (p<0.05). Median time to morbidity was significantly decreased in all treatment arms as compared with controls: vehicle = 28 days, DMXAA = 32 days, anti-PD-1 ab = 39 days, and combination DMXAA + anti-PD-1 ab = 53 days (p<0.05). Combination therapy resulted in significantly longer overall survival than single agent therapy (DMXAA vs. DMXAA+anti-PD-1 ab, p=0.032; anti-PD1 ab vs. DMXAA+antii-PD-1 ab p=0.038)(n=total 13-16 mice per group) (representative data shown in Figure 1). Therapy with DMXAA alone and in combination with anti-PD-1 ab was associated with markedly higher PD-1, PD-L1, and PD-L2 expression levels in bone marrow cells harvested from leukemic mice 48h after treatment. Significantly higher numbers of activated T cells were also identified in the bone marrow and spleen of leukemic mice following two weeks of DMXAA therapy alone or in combination with anti-PD-1 ab. Additional in vivo measurements of systemic cytokine levels following therapy are underway.Conclusions: Here we demonstrate that the combination of an innate immune agonist (DMXAA) with an immune checkpoint inhibitor (anti-PD-1 ab) improved anti-leukemic effects in a preclinical AML model. In vitro DMXAA therapy inhibited murine AML growth in a dose dependent manner, enhanced PD-L1 expression, and induced leukemic production of cytokines (IL-6, IFN-β). In vivo combination DMXAA and anti-PD-1 ab therapy in an immunocompetent murine AML model increased activated host T cell numbers and marrow PD-1/L1/L2 expression in conjunction with decreased tumor burden and prolonged overall survival. These studies may pave the way for future clinical trials evaluating this novel immunomodulatory strategy in AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate.The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML.In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs.The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells.To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells.Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo.Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation.Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. DisclosuresNo relevant conflicts of interest to declare.

The Dose-Dependent Effects of Microrna-155 in Acute Myeloid Leukemia

MicroRNAs are a class of non-coding, regulatory RNAs that control several critical cellular processes. Subsets of microRNAs are dysregulated in cancer, and can act as oncogenes or tumour suppressors.MicroRNA-155 (miR-155) has a well-established role as an oncogene in B cell lymphoma but has a more enigmatic role in acute myeloid leukemia (AML), in which there is evidence that miR-155 may promote or repress the development and progression of AML. We have used enforced expression of miR-155 in murine AML cell lines and AML models to explore the biology of miR-155 in AML.We show that the capacity of miR-155 to promote or repress the ability of AML cells to form colonies and to proliferate is dependent on miR-155 expression levels. Enforced high expression of miR-155 in AML cell lines results in reduced proliferation and colony formation. However, critical long-term assays of cells transduced with miR-155 resulted in selection in favour of an intermediate miR-155 expression level accompanied by a restoration in clonogenic potential. In vivo, enforced expression of miR-155 in murine AML models showed no differences in disease latency compared to controls, but resulted in an increased tumour burden. Most interestingly, RNA-Sequencing analysis demonstrated that the contrasting levels of miR-155 regulate a substantially different set of gene targets, with downstream consequences on transcription that are consistent with the contrasting effects of high and intermediate miR-155 levels. The intermediate levels of miR-155 we observe are the same as that seen in human AML, whereas the high levels of miR-155 have a completely different inflammatory counterpart. Our data shows that that the levels of miR-155 powerfully influences that gene targets it controls and the resultant phenotypes observed. MiR-155 expressed within a specific range promotes AML disease progression. DisclosuresNo relevant conflicts of interest to declare.

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Sensitizing acute myeloid leukemia cells to induced differentiation by inhibiting the RIP1/RIP3 pathway.

Tumor necrosis factor-α (TNF-α)-induced RIP1/RIP3 (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3)-mediated necroptosis has been proposed as an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

Targeting Innate and Adaptive Immune Responses for the Treatment of Acute Myeloid Leukemia

Abstract Background: Successful immunotherapeutic approaches for acute myeloid leukemia (AML) have yet to be developed. We hypothesized that targeting both the innate and adaptive immune responses in leukemic hosts would elicit significant anti-tumor activity with lesser toxicities than chemotherapy. To test this, we evaluated the efficacy of immune checkpoint inhibition (murine anti-PD-1 antibody (ab)) alone and in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an innate immune agonist and anti-vascular agent, in an immunocompetent model of murine AML. Methods: Expression of PD-L1 was assessed by flow cytometry on the murine AML cell line, C1498, alone and following treatment with vehicle, DMXAA or interferon-gamma (positive control). A LEGEND MAX mouse ELISA kit was utilized to measure IL-6 and IFN-β. C57BL/6 mice were inoculated with stably transfected C1498 murine AML cells expressing luciferase and the fluorescent protein DSRed2. Once disease was established, animals were treated with vehicle, DMXAA (20 mg/kg every four days x 7 weeks), anti-murine PD-1 antibody (10 mg/kg every 3 days x 4 doses) or DMXAA + anti-PD-1 antibody (same doses). Animals underwent weekly clinical assessments, weights, and bioluminescent imaging for disease burden. Overall study endpoints were time to morbidity and differences in leukemia disease burden as compared with vehicle-treated controls. Mice were euthanized on day 15 after injection of C1498 cells (8 days following treatment) for collection of plasma, bone marrow, liver and spleen samples for tumor burden, activated T-cells. Results: DMXAA doses (ranging from 1-100 μg/ml) inhibited C1498 in vitro cell growth at 48 hours (48h) in a dose dependent manner. Treatment of C1498 cells in culture with escalating doses of DMXAA (1-100μg/ml) or IFN-gamma (positive control) induced higher PD-L1 expression on these AML cells consistent with direct immunomodulatory effects. Furthermore, C1498 cells exposed to higher doses of DMXAA (10-100μg/ml) for 48h produced measurably higher levels of IL-6 and IFN-β expression in cell supernatants. We then examined the effects of DMXAA, anti-PD-1 ab, or the combination of DMXAA + anti-PD-1 ab treatment in vivo in C57BL/6 mice systemically engrafted with C1498-luciferase AML cells. Treatment overall was well tolerated and resulted in significantly decreased disease burden as measured by total body bioluminescence vs. vehicle controls (p Conclusions: Here we demonstrate that the combination of an innate immune agonist (DMXAA) with an immune checkpoint inhibitor (anti-PD-1 ab) improved anti-leukemic effects in a preclinical AML model. In vitro DMXAA therapy inhibited murine AML growth in a dose dependent manner, enhanced PD-L1 expression, and induced leukemic production of cytokines (IL-6, IFN-β). In vivo combination DMXAA and anti-PD-1 ab therapy in an immunocompetent murine AML model increased activated host T cell numbers and marrow PD-1/L1/L2 expression in conjunction with decreased tumor burden and prolonged overall survival. These studies may pave the way for future clinical trials evaluating this novel immunomodulatory strategy in AML patients. Download : Download high-res image (50KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

Epigenetic therapy as a novel approach for GFI136N-associated murine/human AML.

Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells invitro and invivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted.

Pkc Epsilon Regulates Mitochondrial Redox Biology to Support the Differentiation Blockade in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by the clonal expansion of myeloid progenitors that have impaired differentiation capacity. Determining the molecular machinery that regulate the survival and differentiation blockade of AML cells could serve as a foundation for designing novel therapies. PKCε is a serine-threonine kinase belonging to the subgroup of the Protein Kinase C family called Novel PKCs. Aberrant PKCε expression and activation is associated with the pathogenesis and chemotherapy resistance of many solid cancers. However, the contribution of PKCε in blood malignances such as AML is not well defined.To evaluate the role of PKCe in AML biology, we employed short hairpin RNA (shRNA)-mediated approaches to down-regulate PKCε expression in human and murine AML cell lines. We found that shRNA-mediated knockdown of PKCε significantly reduces the in vitro expansion of several human AML cell lines (MOLM-14, NOMO-1, OCI-AML3, THP-1 and U937). We also observed that blocking PKCε induces caspase-3 cleavage and increases the number of annexin V-positive cells (P<0.05), suggesting that PKCε antagonizes AML cell apoptosis. Additionally, we have also found that prior to cell death, AML cells expressing PKCε-targeting shRNAs display characteristics of myeloid differentiation. Specifically, down-regulation of PKCε results in altered expression of the myeloid differentiation transcription factors C/EBPa and PU.1 and increased expression of the mature myeloid marker CD11b (P<0.001). Moreover, upon PKCε inhibition, AML cells acquire morphological changes associated with differentiation, such as increased cytoplasmic volume, granule formation and nuclear segmentation.Interestingly, we observed similar phenotypic changes when we inhibited PKCε expression in murine AML cell lines driven by the leukemogenic fusion protein MLL-AF9 alone (MLL-AF9) or in combination with the internal tandem duplication mutation of murine Flt3 (MLL-AF9;Flt3-ITD). Specifically, we observed that PKCε down-modulation significantly reduces murine AML cell survival (P<0.001) and colony formation in methylcellulose (P<0.001) of both MLL-AF9 and MLL-AF9;Flt3-ITD cells compared to non-targeting shRNA-expressing cells. We are currently investigating how PKCε inhibition impacts AML progression in vivo using mouse models of AML driven by MLL-AF9 or MLL-AF9;Flt3-ITD.At the molecular level, we have found that PKCε is a key regulator of reactive oxygen species (ROS) biology in AML cells. Specifically, using a fluorogenic probe (CellRox) that indiscriminately detects most types of ROS, we have observed that PKCε knockdown in human AML cell lines results in increased steady-state levels of intracellular ROS compared to shRNA control cells (P<0.002). Total intracellular ROS levels are influenced by the production and clearance of distinct ROS types in various cellular compartments. To further characterize the localization and specific type(s) of ROS regulated by PKCε, we utilized four redox-sensitive GFP (roGFP) probes, which allow for direct measurement of cytoplasmic and mitochrondrial glutathione redox potential (Grx1-roGFP-Cyto and Grx1-roGFP-Mito, respectively) and hydrogen peroxide (H2O2) levels (Orp1-roGFP-Cyto and Orp1-roGFP-Mito, respectively) in live cells. Down-modulation of PKCε in NOMO-1 and OCI-AML-3 cells expressing each of these roGFP constructs resulted in a significant increase in the oxidation of Grx1-roGFP (P<.0007) and Orp1-roGFP-Mito (P<0.02) but not either of cytoplasmic constructs, suggesting that PKCε regulates the production of ROS in the mitochondria of AML cells. Since increased H2O2 production and glutathione oxidation results from increased superoxide (O2-) production in mitochondria, we next evaluated the impact of PKCε on O2- production. Using a fluorogenic probe (MitoSOX) that specifically detects O2- in live cells, we found that PKCε down-modulation increases the production of O2- in AML cells (P<0.05). Our future studies are focused on determining the precise molecular events that connect alterations in redox biology with the survival and differentiation of AML cells.Collectively, these results uncover the previously unrecognized role of PKCε as a critical regulator of mitochondrial redox biology and supporter of cell survival and impaired differentiation in AML. DisclosuresNo relevant conflicts of interest to declare.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008).The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively.The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. [Display omitted] DisclosuresZhang:Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

MUC-1 Regulates MiR34a Expression in Acute Myeloid Leukemia Cells Resulting in an Accumulation of Granulocytic Myeloid-Derived Suppressor Cells

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu that blunts effector cell function and the generation of tumor specific immunity. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppressive tumor microenvironment that fosters immune tolerance and disease growth. The role of MDSCs in AML and the mechanism by which tumor cells evoke the expansion of MDSCs has not been well elucidated. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. The C-terminus of MUC1 (MUC1-C) forms dimers and translocates to the nucleus where it mediates downstream signaling. The effect of MUC1-C mediated signaling on immune modulation in AML has not been well characterized. In prior studies, we have demonstrated that in vitro expansion of MDSCs is correlated with MUC1 expression by the AML cells. In the present study, we sought to characterize the effect of MUC1 on in vivo recruitment of MDSCs and examine the mechanism by which this is accomplished.The murine AML cell line TIB-49 was transplanted in C57BL/6J mice. Following establishment of disease, the mice were euthanized, along with healthy controls, and bone marrow and spleen CD11b+ Gr1+ MDSCs were quantified by flow cytometry. Engrafted mice had an average MDSC burden of 47% in the marrow and 8.7% in the spleen, compared with 35% and 2% in control mice, respectively. The increase in MDSCs was granulocyte predominant, consistent with our findings in patients with AML. Gr1+CD11b+ cells derived from the marrow and spleens of engrafted mice showed higher levels of Arginase-1 compared to MDSCs from control mice, suggesting an increase in immune suppressive phenotype.To investigate the role of MUC1 on the expansion of MDSCs, expression of MUC-1C, the signaling C-terminus of MUC1, was silenced in TIB-49 cells by stable expression of a MUC1-C shRNA as determined by Western Blot analysis. MUC1 silenced or control vector transduced TIB-49 cells were transplanted into C57BL/6J mice. Following establishment of disease, the mice were euthanized, and bone marrow and spleens were quantified for MDSCs. Control AML engrafted mice had an average splenic MDSC burden 2-fold higher than MUC1 silenced AML engrafted mice (n=4). We have developed a cell-penetrating peptide (GO-203) that disrupts homodimerization of the MUC1-C subunit necessary for its downstream signaling. C57BL/6J mice were challenged with TIB-49 AML cells and after 24 hours were treated daily with GO-203. Mice treated with the MUC1 inhibitor had a 2-fold decrease in splenic MDSCs, compared to control mice (n=3) at time of analysis following disease establishment.Noncoding RNAs have emerged as a critical biologic effector of oncogenic pathways. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. miR-34a has been implicated in regulating the expansion of MDSCs. In the present study we demonstrated that silencing of MUC1 expression via lentiviral transduction with a MUC1 specific shRNA resulted in a significant increase in miR-34a expression, as quantified by q-PCR. Consistent with the MUC1 mediated regulation of MDSC expansion by modulation of miR-34a levels, MDSCs induced by co-culture of healthy donor PBMCs with MUC1 silenced AML cells contained 4-fold higher levels of miR-34a, as compared to controls. AML cells were pre-incubated with SYTO® RNASelect™ Green Fluorescent cell stain. After 4 hours, co-cultures of PBMCs and AML cells were analyzed via flow cytometry. AML cells were excluded and cells positive for MDSC markers and containing green fluorescing exosome dye were quantified. AML derived exosomes were found in 18% of MDSCs expanded from AML cells (n=3), demonstrating exosome trafficking from tumor to MDSCs. Finally, miR-34a was over-expressed in MOLM-14 using lentiviral transduction. Over-expression of miR-34a in MUC1 expressing MOLM-14 cells resulted in a 30% reduction in MDSC expansion in co-cultured PBMCs (n=3).In conclusion, MUC1 regulates MDSC expansion in AML, via its effects on miR34a, acting as a critical mediator of tumor mediated immune suppression. Incorporating strategies to reverse the expansion of MDSCs in AML, potentially by targeting MUC1 and increasing miR-34a expression offers a novel therapeutic approach for cancer immunotherapy. DisclosuresKüfe:Genus Oncology, LLC: Equity Ownership.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

Abstract A10: Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model.

Abstract Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Until recently, the tumor-derived signals which stimulate host type I IFN production have remained elusive. However, emerging data suggest that tumor-derived DNA is sufficient to induce IFN-β production in responding cells through activation of a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes). STING-mediated IFN-β production, in turn, leads to functional priming of antigen-specific T cells which mediate tumor rejection in pre-clinical solid tumor models. However, because of their disseminated growth pattern, hematological cancers, such as acute leukemia, may not release sufficient quantities of DNA upon apoptosis to activate the STING pathway in innate immune cells, such as dendritic cells (DCs). The failure of acute leukemias to induce a type I IFN response may contribute to their ability to generate T cell tolerance in the host. Thus, we hypothesized that activating the STING pathway would promote a type I IFN response sufficient to facilitate DC-mediated priming of leukemia-specific T cells and to prolong survival of mice with acute myeloid leukemia (AML). Murine C1498 AML cells engineered to express a model peptide antigen called SIY (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which were then treated with a selective murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control. Preliminary experiments confirmed that DMXAA administration resulted in robust IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control-treated spleen cells). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control-treated animals) of endogenous SIY antigen-specific T cells as determined by SIY/Kb pentamer analysis. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated, leukemia-bearing animals, revealed that the expanded SIY-specific CD8+ T cells were functionally active, and produced high-levels of IFN-γ. To determine if this enhanced immune response translated to a benefit in survival, C1498.SIY AML cell-challenged mice were treated with a single dose of DMXAA or vehicle control five days after tumor inoculation. Among mice treated with DMXAA, 80% of mice rejected the leukemia and survived long-term, while control-treated mice succumbed to AML within approximately 3 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting the DMXAA treatment led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to enhanced survival compared to control-treated animals. This effect was T and or B cell-dependent, as DMXAA treatment of RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway upstream of type I IFN potently enhances adaptive immunity to AML which culminates in prolonged survival or even disease cure in treated animals. It will be important to determine whether the STING pathway may be successfully manipulated in other murine leukemia models. If so, then STING may be a promising therapeutic target to enhance endogenous immunity in AML patients. Citation Format: Emily K. Curran, Xiufen Chen, Leticia Corrales, Justin Kline. Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A10.

Abstract 3216: Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line

Abstract In acute myeloid leukemia (AML), clonal heterogeneity at diagnosis and the presence of minor sub-clones were shown to be responsible for minimal residual disease and relapses in patients. Several studies have proposed that their persistence could be due to their dormant state (i.e. quiescence and/or control of their growth by immune cells). As contribution of immunity to clonal persistence could not be evaluated in xenografts models, we assessed for the clonal diversity of a murine AML cell line. Genomic analyses revealed heterogeneity among the parental cell line and its derived clones. Injected individually into immune-deficient mice, all clones induced AML with different kinetics. However, when administered into immune-competent animals, some clones triggered AML with no mice survival, whereas others elicited reduced lethality rates. The leukemia-surviving mice presented an efficient anti-tumoral CTL activity and reduced lymphopenia. Investigating for residual leukemic cells in the organs of these mice, elimination rather than persistence was observed. Thus, we showed that genetic heterogeneity among the clones led to differential immunogenicity resulting either in their immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients. Citation Format: Carine Brinster, Virginie DRISS, Bruno Quesnel, Martin Figeac, Frédéric Leprêtre, Céline Villenet, Isabelle Briche. Immunogenicity and genomic profiling reveal sub-clonal diversity of a murine acute myeloid leukemia (AML) cell line. [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 3216. doi:10.1158/1538-7445.AM2015-3216

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-induced mutations to the 5’ exons of candidate genes1–5, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We show that the magnitude of negative selection reports the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

Sensitizing Acute Myeloid Leukemia Cells to Interferon-Induced Differentiation By Inhibiting RIP1/RIP3 Necroptotic Pathway

Background: In addition to inducing apoptosis, tumor necrosis factor-α (TNFα) and FAS-induced death receptor signaling also induce necroptosis, a recently defined type of programed necrosis, through activation of the RIP1/RIP3-kinase pathway. The role of death receptor signaling-induced apoptosis in the development of leukemia has been well documented. However, the role of necroptotic signaling in the pathogenesis of leukemia has not been studied.Methods: RIP1/RIP3 signaling was inactivated in murine and human acute myeloid leukemia (AML) cells by genetic deletion of RIP1/RIP3 or pharmacologic inhibition using the specific inhibitor Necrostatin-1. The effects of RIP1/RIP3 signal inactivation on the behavior and interferon-γ (IFNγ)-induced differentiation of AML cells were evaluated by morphologic analysis, cell surface markers, in vitro colony-forming ability and in vivo transplantation/leukemogenic capacity. The responses of mice transplanted with Rip1-/- or Rip3-/-murine AML to IFNγ treatment were compared to the mice transplanted with wild-type AML.Results: We found that most types of AML cells produce TNFα and already show basal level activation of RIP1/RIP3 signaling but they do not undergo necroptosis. Using both pharmacological inhibition and genetic deletion techniques, we determined that inactivation of the RIP1/RIP3 signal resulted in partial differentiation of both human AML cell lines and MLL-AF9-transduced murine AML cells as demonstrated by studies of morphology, cell surface markers and colony-forming ability. Rip1 or Rip3 deletion significantly repressed the leukemogenic capacity of murine AML cells in vivo. In addition, inactivation of RIP1/RIP3 signaling significantly enhanced the complete differentiation of AML cells induced by IFNγ. The combination of RIP1/RIP3 inactivation and IFN treatment significantly further attenuated the clonogenic capacity of both primary AML cells and AML cell lines. Such combination treatment also further compromised the leukemogenic ability of AML cells in vivo. Mechanistically, we found that the RIP1/RIP3 pathway regulates the expression of SOCS1, a key negative regulator of IFNγ signaling. Inactivation of RIP1/RIP3 signaling in AML cells renders them hypersensitive to IFNγ -induced monocytic lineage maturation.Conclusion: Resistance to apoptosis is one of the key mechanisms involved in the development of drug resistance in leukemic cells. RIP1/RIP3-mediated necroptosis induced by TNFα has been proposed to be an alternative therapeutic strategy to treat leukemia. However, our studies suggested that inhibition of RIP1/RIP3-mediated necroptotic signaling might be useful strategy for AML treatment when combined with IFNγ. DisclosuresNo relevant conflicts of interest to declare.