ALBERT

Description: Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL subtypes with distinct genomic features and outcome: adult AEL with bi-allelic alterations in TP53 (31%), frequently co-occurring with alterations in DNMT3A,BCOR, EZH2, RB1, or NFIX; NPM1-mutated (12%); KMT2A-mutated/rearranged (11%), mostly co-mutated with STAG2; pediatric, NUP98-rearranged (5%) and adult, DDX41-mutated (3%). Thirty-eight percent of cases lacked an identifiable exclusive recurrent founding alteration but were enriched in mutations in ASXL1, TET2 and splicing factors. Methods. To explore the roles and cooperativity of the identified alterations in leukemogenesis we used CRISPR-Cas9 genome editing to induce combinations of loss-of-function mutations in 9 recurrently mutated genes in AEL (Tp53, Tet2, Dnmt3a, Asxl1, Ezh2, Stag2, Bcor, Ppm1d, Rb1 and Nfix). Based on patterns of mutation association, we generated 6 pools of lentiviral vectors (Table 1) with different combinations of single guide RNA (sgRNA) to induce multiplex genome editing in Cas9-eGFP-mouse lineage-negative hematopoietic stem cells (HSCs). Transduced cells were transplanted into lethally irradiated congenic mice. Tumors were characterized by morphology, immunophenotyping, and genomic analysis (sequencing of sites of editing, and exome, methylation and transcriptome sequencing). Ex vivo drug screening was performed to test sensitivity to 192 therapeutic agents, including conventional chemotherapeutic agents, and compounds targeting epigenetic regulators, protein kinases and cell cycle checkpoints. Results. In contrast to the uniform representation of guide RNAs observed in HSCs pre-transplant, tumors exhibited enrichment of specific sgRNAs with tumors of specific phenotype. We frequently observed bi-allelic editing of Tp53, Bcor, Tet2, Dnmt3a, Rb1 and Nfix in agreement with the presence of bi-allelic loss in patients. Concomitant editing and inactivation of Tp53/Bcor/Dnmt3a, or Tp53/Bcor/Rb1/Nfix promoted the development of acute erythroid leukemia (GATA1+/RUNX1+/GlyA+/-Ter119+/- and B220/CD19/PAX5/CD3/Mac1/Gr1-). Concomitant editing of Tp53/Bcor/Tet2 promoted the development of B-lineage ALL, and editing of Dnmt3a and Tet2 without Tp53 promoted T-cell ALL. Leukemic clones from primary tumors were serially transplantable across multiple different genetic backgrounds, with the same dominant clone present in all transplanted mice. Notably, mice that did not develop leukemia showed enrichment of different combinations of sgRNAs for Tet2, Dnmt3a and/or Asxl1, genes commonly mutated in clonal hematopoiesis of indeterminate potential, confirming that these mutations are alone not sufficient to induce leukemogenesis. Additional somatic mutations were acquired during clonal expansion of leukemic cells such as alterations of Notch1 and Ikzf1 in T-ALL; Setd2 at the serial passaging of T-ALL; Ptpn11, Kit (D816V), Kras (Q61L) and Lmo7 in the AEL models; and Sf3b3 in the B-ALL model (Fig 1A). Tumors with mutated Tp53 acquired aneuploidy whereas Tet2-mutated cells were genomically stable (Fig. 1B). Unsupervised hierarchical clustering of gene expression profiling identified 3 subgroups (Fig. 1C) associated with distinct genotypes and methylation profiles (Fig. 1D). Tet2-mutated tumors showed increase of hypermethylated sites and co-mutated Bcor/Dnmt3a leukemic cells showed loss of methylation. Eleven tumors representative of key AEL genotypes from the established models were selected to explore drug sensitivity. Response to individual drugs was associated with genotype with co-mutated Tet2/Dnmt3a T-ALL cells sensitive to bromodomain and histone methyltransferase inhibitors; co-mutated Bcor/Tp53/Dnmt3a or Bcor/Rb1 AEL cells to CDK9 inhibitor (LY2857785); Tp53 mutations alone were exclusively sensitive only to PARP inhibition (Talazoparib). In vivo mouse studies are ongoing to confirm ex vivo results. Conclusions: We successfully generated genetically defined models of AEL, demonstrated the role of Tp53 and Bcor mutations in driving the erythroid phenotype, and showed that sensitivity to different classes of compounds is genotype-dependent. These results provide the rational for testing new targeted agents in AEL. Disclosures Mullighan: Abbvie: Research Funding; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Description: Key Points TLI/ATS/alkylator conditioning allows engraftment without GVHD after curative MHC-mismatched BMT for murine β-thalassemia. Recipient MDSCs generated in TLI/ATS/alkylator nonmyeloablative conditioning facilitate donor Treg recovery and graft-versus-host tolerance.

Description: Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied. In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p

Description: The marine environment has been shown to be a rich source of pharmacologically-active secondary metabolites. Three marine- compounds have FDA approval for cancer indications. Aaptamine is a sponge-derived alkaloid that exhibits multiple pharmacological activities including proapoptotic/antiproliferative effects on leukemia cell lines. The effect of the aaptamine class has not been previously studied for high risk leukemias with mixed-lineage leukemia (MLL) gene rearrangements. Using the CellTiter-Glo cell viability assay we evaluated the cytotoxic effect of aaptamine against a panel of leukemia cell lines. We observed that cell lines containing t(4;11) are the most sensitive to aaptamine. Translocation (4;11) is associated with mixed-lineage leukemia and responsible for a very aggressive and refractory pediatric leukemia. Specifically, infants less than one year with t(4;11) have poor survival rates (≈ 19%) and new therapies are urgently needed. Interestingly other MLL cell lines that contain t(9;11) are comparatively less susceptible to aaptamine-mediated cytotoxicity. Jurkat cells overexpressing MLL-AF4 fusion protein are also more sensitive to aaptamine-induced cytotoxicity than wild type or MLL-AF9 overexpressing Jurkat cells indicating the specificity of aaptamine for t(4;11). To further confirm the specificity we conducted a flow based apoptosis assay and observed that aaptamine induces significant apoptosis and necrosis in RS4;11 and MV4;11 cell lines starting at 10µM but not in the t(9;11) containing THP1 cell line. We also found that aaptamine treatment induced G0/G1 arrest specifically in t(4;11) containing cell lines but not in THP1. Additionally we observed that aaptamine did not induce any resistance to the sensitive cell lines after 27 days of chronic exposure. Importantly the compound was well tolerated by healthy activated PBMCs and mice at high concentrations. In order to decipher the mechanism of specificity, we conducted a global proteomic study with treated and untreated RS4;11 and THP1 cell lines. Our proteomic data revealed a significant upregulation of p21 and p27 in aaptamine treated RS4;11 cells but not in THP1. In agreement with the proteomic data, we observed a dose-dependent upregulation of p21 and p27 in both protein and mRNA levels in RS4;11 and MV4;11 cells but not in resistant THP1 cells. Using p21 and p27 promoter-driven luciferase reporter constructs, we observed a significant upregulation of luminescence signal in the RS4;11 cell line at much lower concentration of aaptamine (1µM) whereas the THP1 cell line required 50µM of aaptamine for significant increase in luminescence signal. Cyclin-dependent kinase regulates the G1/S cell cycle transition by phosphorylating retinoblastoma protein (RB). Upregulation of cyclin-dependent kinase inhibitors, such as p27 and p21, promote RB hypophosphorylation and induce G0/G1 arrest. To confirm that this molecular mechanism is responsible for aaptamine induced G0/G1 arrest, we investigated the effect of aaptamine on Rb phosphorylation. We observed a dose dependent downregulation of Rb phosphorylation by aaptamine in sensitive cell lines and predicted it as a major cause of cell cycle arrest. Previous studies have shown that translocation (4;11) is associated with p27 upregulation; thus we hypothesize by further upregulating p27, aaptamine may induce G0/G1 arrest specifically in t(4;11) containing cell lines. To validate the efficacy of aaptamine in vivo, we xenografted 10 NSG mice with 1 million luciferase expressing RS4;11 cells. Four days after leukemia induction we treated half of the mice with subcutaneous injection of aaptamine (100mg/kg, daily) and the other half received vehicle treatment. Bioluminescence imaging (BLI) data revealed a significantly lower disease (p〈 0.03) burden in the aaptamine treated group compared to vehicle treated group after 2 weeks. These findings are being confirmed in patient samples. Additional aaptamine analogs are being designed and will be evaluated for improved therapeutic efficacy. Together our in vitro and in vivo findings suggest that by inducing p21 and p27 aaptamine can induce cell cycle arrest and eventually apoptosis specifically in leukemia cells that contain t(4;11) with relatively low toxicity . Therefore the aaptamine class of drug may provide additional therapeutic options for t(4;11) containing high-risk MLL leukemia patients. Disclosures No relevant conflicts of interest to declare.