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

“Transplantation studies of human leukemias demonstrated that leukemia initiating capacity is highest in CD34+CD38 stem cells (Bonnet and Dick, 1997).”

“Gene set enrichment analysis (GSEA) analysis showed that VTet2 / Flt3ITD LSK cells were characterized by enrichment of an embryonic stem cell signature and reduced expression of a GATA target signature (Figure 4C) (Liberzon et al., 2011; Wong et al., 2008).”

“TET2 catalyzes the 5-hydroxylation of methylcytosine to 5-hydroxymethylcytosine (5-hmC) leading to DNA demethylation (Guo et al., 2011; Tahiliani et al., 2009).”

“Although the combination of Tet2 and Flt3 mutations in our mouse model was sufficient to promote leukemogenesis, additional mutations and chromosomal alterations are sometimes seen in TET2;FLT3ITD mutant human AML (Cancer Genome Atlas Research Network, 2013), suggesting additional events may be required to transform h…”

“Analysis of AML and normal subjects suggests mutations in TET2 and in epigenetic regulators can represent early acquiredmutations in ‘‘pre-leukemic’’ stem cells, which subsequently acquire further mutations leading to overt transformation (Genovese et al., 2014; Jaiswal et al., 2014; Jan et al., 2012).”

“…of AML and normal subjects suggests mutations in TET2 and in epigenetic regulators can represent early acquiredmutations in ‘‘pre-leukemic’’ stem cells, which subsequently acquire further mutations leading to overt transformation (Genovese et al., 2014; Jaiswal et al., 2014; Jan et al., 2012).”

“In addition, mutations in IDH1 and IDH2 inhibit TET2 function through production of 2-hydroxyglutarate (Figueroa et al., 2010), and WT1 mutations lead to reduced TET2 function and reductions in 5hmC (Rampal et al.”

“Notably, of the 27 genes with promoter DMRs and differential expression, 19 were differentially methylated in TET2 mutant AML patients (Table S6) (Akalin et al., 2012a).”

“In particular, TET2mutations are associated with adverse outcome in the setting of concurrent FLT3ITD mutations (Hou et al., 2014; Patel et al., 2012), suggesting thesemutations cooperate to induce a poor-prognosis subtype.”

“Recent studies have also demonstrated the biologic and clinical relevance of these alleles, including TET2, a gene targeted by mutations in 10%–20% of AML patients (Abdel-Wahab et al., 2009; Delhommeau et al., 2009; Langemeijer et al., 2009).”

“classified broadly as those acting on proliferation or on hematopoietic differentiation (Gilliland and Griffin, 2002).”

“Next-generation sequencing has been used to characterize the AML genome and to identify somatic disease alleles (Cancer Genome Atlas Research Network, 2013; Welch et al., 2012).”

“In the murine context, transplantation studies using MLL-fusion leukemia models have shown that GMPs can serve as LSCs, with acquisition of self-renewal by committed progenitors (Heuser et al., 2011; Krivtsov et al., 2006).”

“TET2 catalyzes the 5-hydroxylation of methylcytosine to 5-hydroxymethylcytosine (5-hmC) leading to DNA demethylation (Guo et al., 2011; Tahiliani et al., 2009).”

“TET2 mutant AML is characterized by reduced 5-hmC levels, increased DNA methylation, and epigenetic silencing (Figueroa et al., 2010; Ko et al., 2010; Rampal et al., 2014).”

“Flow Cytometry and Fluorescence-Activated Cell Sorting Antibody staining and fluorescence-activated cell sorting (FACS) analysis were performed as previously described (Moran-Crusio et al., 2011).”

“This may include combination therapies that incorporate epigenetic modulators such as hypomethylating agents and bromodomain inhibitors, or targeted therapies such as the mutant IDH1 and IDH2 inhibitors (Rohle et al., 2013; Wang et al., 2013).”

“In mouse models, conditional loss of Tet2 in the hematopoietic compartment leads to expansion of stem cells (LSK, lin Sca+cKit+) and increased repopulation ability in competitive transplantation (Li et al., 2011; Moran-Crusio et al., 2011; Quivoron et al., 2011).”

“In the human context, acquired mutations in TET2 and in genes encoding other epigenetic modifiers (e.g., DNMT3A, ASXL1) are observed in subjects with clonal hematopoiesis without evidence of an overt hematologic malignancy (Busque et al., 2012; Xie et al., 2014).”

“This may include combination therapies that incorporate epigenetic modulators such as hypomethylating agents and bromodomain inhibitors, or targeted therapies such as the mutant IDH1 and IDH2 inhibitors (Rohle et al., 2013; Wang et al., 2013).”

“Mutations in TET2 lead to loss of function and are often present as heterozygous mutations, although a subset of patients has bi-allelic TET2 loss (Cancer Genome Atlas Research Network, 2013; Langemeijer et al., 2009).”

“FLT3 inhibitors have clinical activity in FLT3ITD mutant AML but do not achieve sustained remission (Kayser and Levis, 2014; Smith et al., 2012).”

“Recent studies have also demonstrated the biologic and clinical relevance of these alleles, including TET2, a gene targeted by mutations in 10%–20% of AML patients (Abdel-Wahab et al., 2009; Delhommeau et al., 2009; Langemeijer et al., 2009).”

“Analysis of AML and normal subjects suggests mutations in TET2 and in epigenetic regulators can represent early acquiredmutations in ‘‘pre-leukemic’’ stem cells, which subsequently acquire further mutations leading to overt transformation (Genovese et al., 2014; Jaiswal et al., 2014; Jan et al., 2012).”

“Gene set enrichment analysis (GSEA) analysis showed that VTet2 / Flt3ITD LSK cells were characterized by enrichment of an embryonic stem cell signature and reduced expression of a GATA target signature (Figure 4C) (Liberzon et al., 2011; Wong et al., 2008).”

“Furthermore, AML profiling has identified somatic GATA2 mutations and dysregulated GATA2 expression (Celton et al., 2014; Cancer Genome Atlas Research Network, 2013; Gröschel et al., 2014).”

“We identify the LSC population in our model of TET2;FLT3 mutant AML in the MPP compartment, a population that has also been found to be expanded in human AML samples and to be capable of leukemia propagation in xenotransplant studies (Goardon et al., 2011).”

“In mouse models, conditional loss of Tet2 in the hematopoietic compartment leads to expansion of stem cells (LSK, lin Sca+cKit+) and increased repopulation ability in competitive transplantation (Li et al., 2011; Moran-Crusio et al., 2011; Quivoron et al., 2011).”

“CpG islands and particularly CpG shores have been described to be critical regulatory regions for hematopoiesis (Ji et al., 2010).”

“Clinical studies indicate increased sensitivity to hypomethylating agents in TET2 mutant patients (Bejar et al., 2014; Traina et al., 2014), which may reflect an ability to reverse the effects of TET2 mutations on DNA methylation by these agents.”

“More recent studies have suggested that leukemic stem cell (LSC) activity can also be detected in committed myeloid progenitors (Kirstetter et al., 2008; Sarry et al., 2011).”

“TET2mutations are associated with reduced response of AML to chemotherapy and adverse overall survival (Metzeler et al., 2011).”

“Clinical studies indicate increased sensitivity to hypomethylating agents in TET2 mutant patients (Bejar et al., 2014; Traina et al., 2014), which may reflect an ability to reverse the effects of TET2 mutations on DNA methylation by these agents.”

“Notably, of the 27 genes with promoter DMRs and differential expression, 19 were differentially methylated in TET2 mutant AML patients (Table S6) (Akalin et al., 2012a).”

“TET2 mutant AML is characterized by reduced 5-hmC levels, increased DNA methylation, and epigenetic silencing (Figueroa et al., 2010; Ko et al., 2010; Rampal et al., 2014).”

“More recent studies have suggested that leukemic stem cell (LSC) activity can also be detected in committed myeloid progenitors (Kirstetter et al., 2008; Sarry et al., 2011).”

“…and 5 kb 50 region) calculated by logistic regression analysis, filtered for R6 DMC. 512 Cancer Cell 27, 502–515, April 13, 2015 ª2015 Elsevier Inc. to the erythroid lineage acquires 5-hmC at binding sites enriched forGATA1andGATA2motifs anddependsonTET2 (Madzoet al., 2014; Pronier et al., 2011).”

“Isolation of human stem and progenitor populations from mononuclear cells was performed as previously described (Barreyro et al., 2012).”

“These data underscore recent studies showing that Idh2;Flt3ITD mutant AML is characterized by a reduced GATA gene expression signature (Kats et al., 2014) and that IDH1 and IDH2mutant AML samples are characterized by differential methylation of GATA target genes (Figueroa et al., 2010).”

“FLT3 inhibitors have clinical activity in FLT3ITD mutant AML but do not achieve sustained remission (Kayser and Levis, 2014; Smith et al., 2012).”

“(legend continued on next page) Cancer Cell 27, 502–515, April 13, 2015 ª2015 Elsevier Inc. 503 out in large AML cohorts has shown that these disease alleles and their combination can be used to improved prognostic schema (Hou et al., 2014; Patel et al., 2012).”

“This is in contrast to reported FLT3 inhibition in Flt3ITD mice where aberrant differentiation is effectively reversed (Chu et al., 2012).”

“In the murine context, transplantation studies using MLL-fusion leukemia models have shown that GMPs can serve as LSCs, with acquisition of self-renewal by committed progenitors (Heuser et al., 2011; Krivtsov et al., 2006).”

“Conditional Vav-creTet2 (VTet2 / ) mice were crossed to the constitutive knockin Flt3 murine model (Lee et al., 2005) to generate Vav-creTet2;Flt3 (VTet2 / Flt3) mice.”

“Epigenetic Silencing of Gata2 Contributes to Impaired Differentiation in VTet2Flt3 LSC The Gata2 locus in VTet2 / Flt3 LSKs was characterized by marked hypermethylation at the promoter, introns, and a key enhancer region compared with WT and single mutant LSKs (Figure 5G) (Snow et al., 2010).”

“Furthermore, AML profiling has identified somatic GATA2 mutations and dysregulated GATA2 expression (Celton et al., 2014; Cancer Genome Atlas Research Network, 2013; Gröschel et al., 2014).”

“AML1-ETO or FLT3-ITD (two common oncogenic alterations) in Tet2-depleted hematopoietic cells results in complete leukemogenic transformation into AML-like diseases (Rasmussen et al. 2015; Shih et al. 2015; Hatlen et al. 2016).”

“CD48 + CD150 − LSK HPC cells, which can initiate AML from Flt3 ITD ;Tet2 Δ/+ cells 31 , expanded to a significantly greater extent in the bone marrow (Extended Data Fig.”

“In a recent published combinatorial model of TET2 knockout and FLT3-ITD mutation, secondary transplantation of FACS-purified LSK CD48(1) CD150(2) MPP-like cells, which are not normally associated with self-renewal, initiated andmaintained leukemia.(69) In contrast, GMPlike blasts were unable to initiate disease, sugge…”