ALBERT

Description: Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

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: Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype characterized by kinase-activating alterations. One recurrent alteration is the ETV6-NTRK3 fusion, which results in constitutive activation of NTRK3, a member of the neurotrophic receptor kinase family. ETV6-NTRK3 has been identified in a range of malignancies, including breast cancer, pediatric glioma and infantile fibrosarcoma. The oncogenic role of ETV6-NTRK3 in B-cell ALL has not been investigated. The goals of this study were to assess the development of leukemia in genetically engineered models of ETV6-NTRK3, and to investigate efficacy of the specific TRK A, B and C inhibitor, LOXO-101, currently in clinical trials for the treatment of solid tumor patients who harbor NTRK fusions. Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vitro and in vivo. Results: Etv6-NTRK3/+, CD19-Cre mice developed aggressive disease with 100% penetrance and a median latency of 38 days (n=27). The average body weight of Etv6-NTRK3/+, CD19-Cre mice was significantly reduced compared to age-matched Etv6-NTRK3/+ controls (13.9 vs 20.2g, p

Description: Background: Osteonecrosis and osteopenia frequently complicate acute lymphoblastic leukemia (ALL) therapy. Interventions which reduce these bony toxicities without impairing the anti-leukemic efficacy of therapy are lacking. Case reports suggest that bisphosphonates may reduce the pain associated with osteonecrosis; however, it is unclear if they are effective in preventing or reducing the progression of osteonecrosis or modifying bone density loss during therapy. Their impact on the anti-leukemic efficacy of therapy is also unknown. Methods: We tested the efficacy of the bisphosphonate zoledronic acid (ZA) in the murine model of osteonecrosis. Four-week-old Balb/c mice were treated with dexamethasone (2mg/L in drinking water) and PEGylated-asparaginase [1200IU/kg intraperitoneal (IP) weekly] for 6 weeks. To test whether ZA could prevent osteonecrosis, mice received ZA 100ug/kg IP weekly starting with therapy initiation (early zoledronic acid group, receiving 6 total doses). To test whether ZA could prevent the progression of osteonecrosis, ZA administration was delayed until the fourth week of therapy (late zoledronic acid group, receiving 2 total doses) after osteonecrosis processes have already begun. All mice were humanly euthanized at 10 weeks post-natal. At the end of the 6 week therapy, osteonecrosis was assessed histologically at the distal femur, while bone density and quality measurements were obtained from the proximal tibia using the Siemeons Inveon uCT system. To test the impact of zoledronic acid on the anti-leukemic efficacy of therapy, nine-week-old B6 mice were injected with 2000 Arf-/- Bcr/Abl+ syngeneic acute lymphoblastic leukemia cells. Mice received three weeks of therapy identical to the osteonecrosis studies (including zoledronic acid randomization), except dexamethasone was increased to 4mg/L in the drinking water. Mice were sacrificed when moribund and leukemia as cause of death was confirmed by peripheral white blood cell count and spleen weight. Results: Osteonecrosis was reduced in the early zoledronic acid group compared to the control group (12/69 limbs vs. 25/72 limbs, p=0.03). The incidence of osteonecrosis in the late zoledronic acid group (19/56 limbs) was identical to that in the control group (p=1). Both zoledronic acid interventions increased the cortical thickness of the tibia [median 0.12mm in therapy treated controls; 0.18mm in early zoledronic acid group (p=

Description: Key Points We established a Nudt15 knockout mouse model with which to evaluate individualized thiopurine therapy. Preemptive NUDT15 genotype–guided thiopurine dosing can effectively prevent drug toxicity without compromising antileukemic efficacy.

Description: Abstract 3560 Thiopurines are routinely used to treat many hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), as well as inflammatory and autoimmune diseases. Common polymorphic null alleles among humans in thiopurine methyltransferase (TPMT) render 1 in 400 patients equivalent to homozygous knock-outs (−/−), 10% of patients equivalent to hemizygous (+/−), and 90% of patients comparable to wild-type (+/+) status for TPMT enzyme activity. TPMT activity is inversely related to the accumulation of active thioguanine nucleotide metabolites among patients, resulting in a higher risk for myelosuppressive adverse effects if routine doses are given to −/− compared to +/− compared to +/+ patients. However, the impact of the host TPMT polymorphism on antileukemic efficacy in the clinic is less clear. We generated a murine model of ALL by transducing bone marrow cells of Arf-null mice on the 129X1SvJ background (Tpmt +/+) with a retroviral vector encoding p185 BCR-ABL and luciferase, and culturing them for 7–10 days. We injected 2000 BCR-ABL+ pro/pre-B cells into Tpmt-deficient (−/−), heterozygous (+/−), or wild type (+/+) unconditioned syngenic recipients and compared the antileukemic efficacy when treating with a relatively low dose of mercaptopurine (equivalent to 7–9 mg/m2—such as would be tolerated by patients homozygous for null TPMT alleles). Treatment was started one day after injection of leukemia cells and continued for 70 days or until mice were sacrificed due to moribund state (usually due to illness from leukemia). To mimic clinically relevant treatment regimens, methotrexate was injected intraperitoneally (IP) at 1 mg/kg, once a week. Mercaptopurine was administered in the drinking water at 10 mg/L, for an estimated delivered dose of 2 – 2.5 mg/kg/day of mercaptopurine. Erythrocyte TPMT activity segregated by Tpmt genotype as expected and was comparable to the differences observed in patients: 0.6 U/ml in Tpmt −/−, 6.7 U/ml in Tpmt +/−, and 11.6 U/ml in Tpmt +/+ mice (p =0.008, n=2 for each genotype). As expected, the median survival (± standard error) was similar among Tpmt genotypes in untreated mice: 16 (± 0.4) days for Tpmt +/+, 17 (± 0.7) days for Tpmt +/−, and 18 (± 0.4) days for Tpmt −/−. The difference in leukemia-free survival by Tpmt genotype was profound: at 30 days after the start of treatment, 5% (± 9%) of Tpmt +/+ mice, 47% (± 26%) of Tpmt +/− mice, and 85% (± 14%) of Tpmt −/− mice were surviving (p = 5×10−8, Figure), indicating a substantial impact of host Tpmt status on thiopurine antileukemic effectiveness. In vivo imaging of luciferase-marked ALL cells confirmed an intermediate response phenotype for the Tpmt heterozygous mice (p = 0.00001). With higher doses (closer to the doses tolerated by patients homozygous for wild-type alleles, equivalent to 50–70 mg/m2/day), leukemia-free survival improved in the Tpmt +/+ and Tpmt +/− mice, comparable to the survival of Tpmt −/− mice treated with the low dose of mercaptopurine. These findings support the notion that germline polymorphisms in Tpmt, even in the heterozygous state, affect not only host tissue toxicity, but antitumor effectiveness as well. Disclosures: Evans: St. Jude Children's Research Hospital: Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms., Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms. Patents & Royalties. Relling:St. Jude Children's Research Hospital: Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms., Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms. Patents & Royalties; Sigma-Tau Pharmaceuticals: Research Funding.

Description: Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia. Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice. Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation. Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.

Description: Introduction: The genetic basis of several acute myeloid leukemia (AML) subtypes remains poorly characterized, such as that of acute erythroid leukemia (AEL, AML M6) which is currently subclassified by morphology alone, and is associated with poor outcome. Here we sought to perform a definitive genomic analysis of AEL and translate these findings into faithful experimental models and novel therapeutic approaches. Methods: We studied 151 AEL cases (19.2% pediatric, 4.6% young adult, 21.9% adult and 54.3% older adult). Diagnosis of AEL was centrally confirmed and subclassified according to WHO 2008 and revised 2016 criteria. Whole exome and/or genome sequencing, RNA-sequencing and SNP array analysis were performed on all cases and in 2 AEL cell lines (TF-1 and Hel). Genomic data were compared to those from non-M6 childhood and adult AML from TARGET (n=192) and TCGA (n=197) studies. The functional effects of fusion transcripts and mutated genes were examined in IL-3 dependent Ba/F3 cells, NIH3T3 cells for focus formation assays and/or mouse lineage negative hematopoietic stem cells (lin- HSC) for colony forming and transplantation assays. Avatars of human AEL were established in immunocompromised NSGS and MISTRG mice for preclinical studies. Results: a) Genomic landscape of AEL. We identified 2,250 non-synonymous clonal and subclonal somatic mutations in 1,723 genes with a mean of 16.4 per case (range 2-88) and with missense and frameshift mutations accounting for 47.1% and 22.5% of all mutations, respectively. 78 genes were recurrently mutated in at least 3 cases. In frame fusions were detected in 31% of childhood and 27.5% of adult cases, and were more frequent in cases with complex karyotype. 124 potential driver genes were identified by statistical analysis or known pathogenic role in cancer, 9 of which were recurrent novel targets of mutation, most commonly involving chromatin modification (60.3%), cell cycle/tumor suppression (TP53, 33.8%), DNA methylation (28.5%), transcription regulation (26.5%), splicing (15.9%), NPM1 (11.9%), Ras (11.3%), JAK-STAT signaling (9.9%), the cohesin complex (8.6%), ALK/NTRK1 (4.6%) and PI3K signaling (3.3%). Overall, 33% of cases harbored a mutation in signaling genes amenable to inhibition by tyrosine kinase/Ras inhibitors. Mutations in TP53 and DNA methylation genes were significantly more frequent in adults while mutations in transcription regulators and Ras pathway were more frequent in children. Splicing mutations correlated with MDS phenotype and PI3K alterations with therapy-related AEL. Based on the co-occurrence and exclusivity of mutations 7 main distinct AEL genetic subtypes were defined: 1) pediatric AEL with NUP98-rearrangements (3.3% of all cases); 2) adult complex karyotype AEL with TP53 mutations (33.8%); 3) AEL with MLL-rearrangements (12.6%); 4) NPM1-mutated AEL (11.9%); 5) DNA-methylation/splicing mutated AEL (17.8%); 6) splicing/Ras/transcription regulation mutated AEL (21.2%) and 7) Other (8.6%) (Fig.1A). Mutations of chromatin modifiers occurred independently of karyotype, age and subtype (Fig.1B). NUP98-fusions and mutations in PTPN11, UBTF and GATA1 were more frequent in pediatric AEL compared to non-M6 AML. Among adults, mutations in TP53 and MLL were more frequent in AEL while FLT3, NPM1, DNMT3A and IDH1 were more in frequent in non-M6 subtypes. A complex karyotype, therapy-related AEL, TP53 mutations and NUP98-rearrangements were associated with poor outcome. b) Functional AEL modeling and therapeutic translations. Expression of NUP98-JARID1A in lin- HSC resulted in sustained self-renewal and development of an aggressive transplantable leukemia. At least three classes of signaling pathway mutations are targetable in AEL. ALK mutations in the extracellular MAM domain transformed Ba/F3 cells which were sensitive to crizotinib in vitro. Mutations in the tyrosine kinase domain of NTRK1 transformed NIH3T3 cells and were sensitive to entrectinib in vitro. Targeting of JAK-STAT, mTOR and PI3K pathways were examined in xenografts and sensitivity to JAK2 inhibitor ruxolitinib was confirmed in vivo. Conclusions: We provided the first comprehensive landscape of genomic alterations in AEL and defined distinct genomic groups with unique patterns of mutation occurrence compared to non-M6 AML. Finally, we showed that several pathogenic pathways are amenable to inhibition by approved targeted compounds. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Wei:Novartis: Honoraria, Research Funding. Loh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Research Funding.