Kaitlyn Shank

Mutational Cooperativity Linked to Combinatorial Epigenetic Gain of Function in Acute Myeloid Leukemia

Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.

Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms

The discovery of JAK2/MPL mutations in patients with myeloproliferative neoplasms (MPN) led to clinical development of Janus kinase (JAK) inhibitors for treatment of MPN. These inhibitors improve constitutional symptoms and splenomegaly but do not significantly reduce mutant allele burden in patients. We recently showed that chronic exposure to JAK inhibitors results in inhibitor persistence via JAK2 transactivation and persistent JAK-signal transducer and activator of transcription signaling. We performed genetic and pharmacologic studies to determine whether improved JAK2 inhibition would show increased efficacy in MPN models and primary samples. Jak2 deletion in vivo led to profound reduction in disease burden not seen with JAK inhibitors, and deletion of Jak2 following chronic ruxolitinib therapy markedly reduced mutant allele burden. This demonstrates that JAK2 remains an essential target in MPN cells that survive in the setting of chronic JAK inhibition. Combination therapy with the heat shock protein 90 (HSP90) inhibitor PU-H71 and ruxolitinib reduced total and phospho-JAK2 and achieved more potent inhibition of downstream signaling than ruxolitinib monotherapy. Combination treatment improved blood counts, spleen weights, and reduced bone marrow fibrosis compared with ruxolitinib alone. These data suggest alternate approaches that increase JAK2 targeting, including combination JAK/HSP90 inhibitor therapy, are warranted in the clinical setting.

DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3AR882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

Combination Targeted Therapy to Disrupt Aberrant Oncogenic Signaling and Reverse Epigenetic Dysfunction in IDH2- and TET2-Mutant Acute Myeloid Leukemia.

Genomic studies in acute myeloid leukemias (AML) have identified mutations that drive altered DNA methylation, including TET2 and IDH2 Here, we show that models of AML resulting from TET2 or IDH2 mutations combined with FLT3ITD mutations are sensitive to 5-azacytidine or to the IDH2 inhibitor AG-221, respectively. 5-azacytidine and AG-221 treatment induced an attenuation of aberrant DNA methylation and transcriptional output and resulted in a reduction in leukemic blasts consistent with antileukemic activity. These therapeutic benefits were associated with restoration of leukemic cell differentiation, and the normalization of hematopoiesis was derived from mutant cells. By contrast, combining AG-221 or 5-azacytidine with FLT3 inhibition resulted in a reduction in mutant allele burden, progressive recovery of normal hematopoiesis from non-mutant stem-progenitor cells, and reversal of dysregulated DNA methylation and transcriptional output. Together, our studies suggest combined targeting of signaling and epigenetic pathways can increase therapeutic response in AML.Significance: AMLs with mutations in TET2 or IDH2 are sensitive to epigenetic therapy through inhibition of DNA methyltransferase activity by 5-azacytidine or inhibition of mutant IDH2 through AG-221. These inhibitors induce a differentiation response and can be used to inform mechanism-based combination therapy. Cancer Discov; 7(5); 494-505. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Yen et al., p. 478This article is highlighted in the In This Issue feature, p. 443.

Dnmt3a regulates myeloproliferation and liver-specific expansion of hematopoietic stem and progenitor cells.

DNA methyltransferase 3A (DNMT3A) mutations are observed in myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Transplantation studies have elucidated an important role for Dnmt3a in stem cell self-renewal and in myeloid differentiation. Here, we investigated the impact of conditional hematopoietic Dnmt3a loss on disease phenotype in primary mice. Mx1-Cre-mediated Dnmt3a ablation led to the development of a lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by peripheral cytopenias and by marked, progressive hepatomegaly. We detected expanded stem/progenitor populations in the liver of Dnmt3a-ablated mice. The MDS/MPN induced by Dnmt3a ablation was transplantable, including the marked hepatomegaly. Homing studies showed that Dnmt3a-deleted bone marrow cells preferentially migrated to the liver. Gene expression and DNA methylation analyses of progenitor cell populations identified differential regulation of hematopoietic regulatory pathways, including fetal liver hematopoiesis transcriptional programs. These data demonstrate that Dnmt3a ablation in the hematopoietic system leads to myeloid transformation in vivo, with cell-autonomous aberrant tissue tropism and marked extramedullary hematopoiesis (EMH) with liver involvement. Hence, in addition to the established role of Dnmt3a in regulating self-renewal, Dnmt3a regulates tissue tropism and limits myeloid progenitor expansion in vivo.

Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity

Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and NrasG12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

Aid is a key regulator of myeloid/erythroid differentiation and DNA methylation in hematopoietic stem/progenitor cells

Recent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation.

JAK2/IDH-mutant–driven myeloproliferative neoplasm is sensitive to combined targeted inhibition

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617F and mutant IDH1R132H or Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617F Idh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mut and IDH2mut mutations. Taken together, these data suggest that combined JAK and IDH inhibition may offer a therapeutic advantage in this high-risk MPN subtype.

Abstract 44: Leukemia-associated DNMT3A R882 mutations and their role in anthracycline-induced DNA damage response and therapeutic resistance

Despite significant advances in cancer research and treatment, therapeutic resistance remains a major obstacle for achieving stable remission in cancer patients. Acute myeloid leukemia (AML) is no exception, and most AML patients develop resistance to chemotherapy/targeted therapies, which results in disease relapse and progression. Recurrent mutations in the DNA methyltransferase 3A (DNMT3A) gene have been identified in 20-30% of AML cases and are predictive of unfavorable prognosis in patients treated with standard anti-leukemic regimens. In addition, DNMT3A-mutant AMLs appear to be relatively refractory to anthracycline family chemotherapeutics, such as daunorubicin. Half of all DNMT3A mutations affect amino acid residue R882, and recent work has shown that these mutants display decreased enzymatic activity and aberrant binding properties. In addition, previous studies have shown that wild-type DNMT3A functions as a pro-apoptotic switch in response to genotoxic stress induced by another anthracycline doxorubicin. We propose that mutant DNMT3A protects cells from apoptosis in response to DNA damage by altering molecular machinery involved in DNA-damage sensing, response and/or repair, through DNA methylation-dependent or independent mechanisms. Specifically, our data show that mutant DNMT3A affects recruitment of DNA repair proteins to chromatin, including aberrant distribution of homologous recombination marker RAD51. We are currently investigating molecular changes in DNA damage response in DNMT3A-mutant cells in vitro and ex vivo, and leukemogenic potential of the mutant Dnmt3a allele in vivo, whether alone or in combination with other cooperating oncogenes. Citation Format: Olga A. Guryanova, Kaitlyn Shank, Luisa Luciani, Evangelia Loizou, Matthew D. Keller, Abby R. Weinstein, Omar Abdel-Wahab, Siddhartha Mukherjee, Stephen S. Nimer, Ross L. Levine. Leukemia-associated DNMT3A R882 mutations and their role in anthracycline-induced DNA damage response and therapeutic resistance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 44. doi:10.1158/1538-7445.CANSUSC14-44