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

SUMMARY

Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and Nras^G12D 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.

Graphical Abstract

Kunimoto et al. show that concurrent Tet2 loss and Nras^G12D expression in hematopoietic cells induces fully penetrant, lethal chronic myelomonocytic leukemia by decreasing negative regulators of MAPK activation. Mouse and human TET2/NRAS double-mutant leukemia show preferential sensitivity to MEK inhibition.

INTRODUCTION

Gene discovery studies have elucidated the somatic mutational landscape of myeloid malignancies (Cancer Genome Atlas Research Network, 2013). These efforts have revealed distinct classes of mutations including genes that control signal transduction pathways and those that regulate epigenetic modifications. These data suggest that specific combinations of leukemia disease alleles commonly co-occur; in some cases functional studies have shown how specific combinations of alleles cooperate in hematopoietic transformation (Shih et al., 2015).

One of the most common genes targeted by somatic loss-of-function mutations in myeloid malignancies is TET2, a member of the TET family whose encoded proteins catalyze the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) leading to DNA demethylation (Tahiliani et al., 2009). TET2 mutations are reported in various myeloid malignancies (Abdel-Wahab et al., 2009; Delhommeau et al., 2009; Jankowska et al., 2009; Langemeijer et al., 2009). Genetic and functional studies have demonstrated TET2 is a key regulator of hematopoietic stem cell (HSC) self-renewal. Conditional loss of Tet2 in mice leads to expansion of hematopoietic stem/progenitor cells (HSPCs, LSK, lin⁻ Sca1⁺ cKit⁺) and enhanced repopulating capacity in vivo (Moran-Crusio et al., 2011; Quivoron et al., 2011). Furthermore, studies of the genetic basis of clonal hematopoiesis have shown that somatic mutations in TET2 and in other genes encoding epigenetic modifiers, including DNMT3A and ASXL1, promote clonal hematopoietic expansion (Busque et al., 2012; Genovese et al., 2014; Jaiswal et al., 2014; Jan et al., 2012; Xie et al., 2014). These data indicate that TET2 mutations can increase the fitness of stem/progenitor cells to promote clonal expansion, but that additional genetic and epigenetic events are required to induce myeloid transformation.

Ras proteins are small GTPases that activate downstream signaling effectors, such as Raf and phosphoinositide 3-kinase (PI3K), thereby transducing signals from activated growth factor receptors. Somatic RAS mutations encode proteins that accumulate in the active GTP-bound conformation (Schubbert et al., 2007). NRAS is the most common target of oncogenic mutations in myeloid leukemia (Bacher et al., 2006). Mice with a hematopoietic tissue specific Nras^G12D allele develop progressive myeloid expansion (Li et al., 2011), and oncogenic Nras can increase the self-renewal of HSCs (Li et al., 2013). These data underscore the critical role of oncogenic NRAS mutations in myeloid leukemogenesis.

Studies have shown that mutations in epigenetic modifiers and signaling factors commonly co-occur in acute myeloid leukemia (AML), including concurrent TET2/IDH/WT1 mutations and NRAS mutations (Papaemmanuil et al., 2016; Patel et al., 2012). Analysis of the clonal architecture in CMML patients suggests TET2/NRAS double-mutant clones expand over time, including at relapse after allogeneic stem cell transplantation (Itzykson et al., 2013). These data suggest TET2 and NRAS mutations can cooperate to promote myeloid transformation and clonal dominance. Although a previous study reported knockdown of Tet2 did not accelerate disease in a Nras^G12D induced transplant myeloproliferative neoplasm (MPN) model (Chang et al., 2014), to date no studies have assessed whether Tet2/Nras mutations expressed in HSPCs can cooperate in vivo. Of equal importance, the role of concurrent disease alleles in mediating the sensitivity to targeted therapies has not been elucidated, particularly whether concurrent mutations affect the responsiveness to MEK targeted therapies.

RESULTS

Tet2 Loss Combined with Nras^G12D Develops CMML-like Disease In Vivo

To determine if expression of TET2 and NRAS^G12D mutations in stem/progenitor cells could cooperate in hematopoietic transformation, we crossed Mx1-Cre;Tet2^f/f (Tet2^−/−) mice (Moran-Crusio et al., 2011) to Mx1-Cre;Nras^G12D (Nras^G12D) mice (Li et al., 2011) to generate Mx1-Cre;Tet2^f/f;Nras^G12D (referred subsequently as Tet2^−/−Nras^G12D) mice (Figure S1A). After somatic recombination of both alleles, Tet2^−/−Nras^G12D mice developed a lethal hematopoietic disease with a median survival of 37.3 weeks (Figure 1A). The survival of Tet2^−/−Nras^G12D mice was significantly worse compared to wild-type (WT) or mice with either disease allele alone (WT, Tet2^−/− and Nras^G12D median survival >80 weeks). Complete blood counts (CBC) performed 16 weeks post recombination showed leukocytosis, monocytosis, and thrombocytopenia in Tet2^−/−Nras^G12D mice (Figure 1B, 1C, and S1B). Flow analysis of the peripheral blood (PB) revealed an increased proportion of Mac1⁺Gr1⁺ and Mac1⁺Gr1⁻ myeloid cells in Tet2^−/−Nras^G12D mice (Figure 1D, S1C and S1D). Tet2^−/−Nras^G12D mice also had increased proportion of cKit⁺ circulating cells (Figure 1E and S1E). Tet2^−/−Nras^G12D mice developed marked splenomegaly not seen with either mutation expressed in hematopoietic cells alone (mean 617 mg, p<0.05; WT 109 mg, Tet2^−/− 212 mg, Nras^G12D 154 mg) (Figure 1F). Pathological examination of the liver, spleen and lung revealed robust infiltration of myeloid cells, including significant destruction of normal spleen and lung architecture in Tet2^−/−Nras^G12D mice (Figure 1G). Immunohistochemical (IHC) examination showed a marked increase in Ki-67⁺ cells in infiltrating lesions, demonstrating mutant myeloid cells are highly proliferative (Figure S1F). In order to seek the cause of death in Tet2^−/−Nras^G12D mice, we performed analyses of moribund mice compared to matched controls. CBC, flow cytometry and pathologic analysis showed similar phenotype as was observed at 16 weeks but with increasing severity of organ infiltration and expansile lesions (Figure S1G–S1L). These data indicate that Tet2 loss combined with Nras^G12D cooperates to promote progressive, lethal CMML in vivo which is not seen with either mutation alone.

Figure 1. Development of CMML-like Disease in Tet2^−/−Nras^G12D Mice.

(A) Kaplan Meier survival curve of WT (n=31), Tet2^−/− (n=69), Nras^G12D (n=24) and Tet2^−/−Nras^G12D (n=46) mice.

(B–C) Peripheral WBC counts (B) and peripheral monocyte percentage (C) at 16 weeks from PIPC injection (WT; n=40, Tet2^−/−; n=64, Nras^G12D; n=39, Tet2^−/−Nras^G12D; n=55).

(D–E) Representative immunophenotype of peripheral blood Mac1/Gr1 (D) and Mac1/cKit (E) at 16 weeks from PIPC injection, with the percentage of each fraction indicated.

(F) Representative images of spleens and bar graph of spleen weights from each group at 16 weeks from PIPC injection (WT; n=7, Tet2^−/−; n=8, Nras^G12D; n=7, Tet2^−/−Nras^G12D; n=7).

(G) Representative images of HE staining of liver, spleen and lung of WT, Tet2^−/−, Nras^G12D, and Tet2^−/−Nras^G12D mice at 1 year from PIPC injection. The scale bars represent 200 μm.

A log-rank test was used for survival statistics. Otherwise, an unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S1.

Combination of Tet2 Loss and Nras^G12D Causes Altered Hematopoietic Differentiation

Since Tet2 loss or Nras^G12D expression leads to dysregulated HSPC differentiation and altered self-renewal in vivo (Li et al., 2013; Moran-Crusio et al., 2011; Quivoron et al., 2011), we performed flow analysis of stem/progenitor and erythroid lineage compartments in these mice 12 weeks following polyinosinic:polycytidylic acid (PIPC) injection. Tet2^−/−Nras^G12D mice showed a significant increase in bone marrow (BM) LSK cells compared to WT or Tet2^−/− mice (mean, 0.39 % versus 0.17 % WT, 0.19 % Tet2^−/−, p<0.01) (Figure 2A and S2A). In addition, we observed a significant increase in the percentage of BM granulocyte-monocyte progenitors (GMPs, Lineage⁻Sca-1⁻c-Kit⁺CD34^hiFcgR^hi) and a trend towards an increase in BM common myeloid progenitors (CMPs, Lineage⁻Sca-1⁻c-Kit⁺CD34^hiFcgR^low) in Tet2^−/−Nras^G12D mice (Figure 2B and S2B). There was no significant difference in the percentage of BM myeloid progenitors (MPs, Lineage⁻Sca-1⁻c-Kit⁺) and long-term HSCs (SLAM-LSK, CD150⁺CD48⁻LSK) (Figure S2C). Tet2^−/−Nras^G12D mice showed a significant expansion in BM multipotent progenitors (MPPs, CD150⁻CD48⁺LSK) compared to WT or Tet2^−/− mice (Figure S2C). Of note, both CD71⁺Ter119⁺ and CD71⁻Ter119⁺ erythroid compartments were significantly decreased, suggesting that Tet2 loss combined with Nras^G12D leads to further impairment of erythroid differentiation (Figure 2C and S2D). Flow analysis of splenocytes revealed a significant increase in the percentage and the absolute number of splenic MPs as CMPs, GMPs, and megakaryocyte-erythroid progenitors (MEPs, Lineage⁻Sca-1⁻c-Kit⁺CD34^lowFcgR^low) and a trend towards an increase in the proportion and the absolute number of splenic LSKs in Tet2^−/−Nras^G12D mice (Figure 2D, S2E, S2F and S2G). Moreover, they demonstrated a higher percentage and absolute number of Mac1⁺Gr1⁻ myeloid cells in the spleen compared to WT or single-mutant mice (Figure 2D, S2H and S2I). These data suggest Tet2 loss and Nras^G12D cooperate to alter hematopoietic differentiation and self-renewal.

Figure 2. Expanded Stem-Progenitor Fraction and Transplantability of Tet2^−/−Nras^G12D Mice.

(A) The percentage of bone marrow LSK fraction at 12 weeks from PIPC injection (n=3 for each group).

(B–C) Representative immunophenotype of bone marrow CMP, GMP and MEP fraction (B) and erythroid progenitor fraction (C) at 12 weeks from PIPC injection, with the percentage of each fraction indicated.

(D) The percentage of spleen myeloid progenitor (Lineage⁻Sca1⁻cKit⁺), CMP, GMP, MEP and Mac1⁺Gr1⁻ fraction at 12 weeks from PIPC injection (n=4 for each group).

(E) Kaplan Meier survival curve of primary splenic cell recipients (upper panel, Nras^G12D; n=10, Tet2^−/−Nras^G12D; n=10) and secondary recipients (lower panel, Nras^G12D; n=7, Tet2^−/−Nras^G12D; n=8).

(F) Representative images of spleens from primary recipients 9 weeks after transplantation (Nras^G12D; n=3, Tet2^−/−Nras^G12D; n=3).

(G) Representative immunophenotype of donor derived Mac1/Gr1 fraction in peripheral blood and spleen from primary recipients 9 weeks after transplantation.

A log-rank test was used for survival statistics. Otherwise, an unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S2.

Tet2^−/−Nras^G12D Cells Can Propagate CMML-like Disease in Transplant Recipients

We sought to determine whether Tet2 loss and Nras^G12D cooperate to augment stem cell function and leukemogenesis. We selected Nras^G12D and Tet2^−/−Nras^G12D donor mice with similar proportion of splenic LSK cells (Figure S2J) and equal number of CD45.2⁺ splenocytes were transplanted into CD45.1⁺ lethally irradiated recipient mice. The recipients of Tet2^−/−Nras^G12D cells had significantly impaired survival compared to recipients of Nras^G12D cells (median survival 10.6 weeks versus >40 weeks Nras^G12D, p=0.0111), and all recipient mice developed a lethal hematopoietic disease similar to the phenotype in primary mice (Figure 2E). In secondary transplants, all recipient mice engrafted with Tet2^−/−Nras^G12D cells developed disease and died within 4 weeks with marked splenomegaly, whereas no recipients of Nras^G12D cells developed disease or splenomegaly (Figure 2E, 2F, and S2K). The PB and spleen had a significantly higher percentage of donor-derived (CD45.2⁺) myeloid cells in Tet2^−/−Nras^G12D recipients compared to Nras^G12D recipients (Figure 2G and S2L), consistent with increased myeloid expansion with Tet2^−/− and Nras^G12D combination. Pathological examination revealed similar infiltration of Ki-67⁺ myeloid cells with significant destruction of normal spleen and lung architecture (Figure S2M). Of note, some Tet2^−/−Nras^G12D recipients showed additional infiltration of myeloid cells into the spinal cord causing paraplegia and more severe BM fibrosis compared to Nras^G12D recipients (Figure S2M). Injection of Tet2^−/−Nras^G12D cells in sublethally irradiated recipient mice induced significant anemia, thrombocytopenia, neutrophilia, monocytosis, marked splenomegaly with myeloid cell infiltration and expansion (Figure S2N–S2S). Sublethally irradiated Tet2^−/−Nras^G12D recipients also developed CMML-like disease and died within 3 weeks, fulfilling the criteria of CMML-like myeloid leukemia (Kogan et al., 2002) (Figure S2T).

To explore the role of combined Tet2^−/−Nras^G12D on HSC self-renewal, we performed competitive repopulation assays. 5×10³ sorted BM LSK cells from WT, Tet2^−/−, Nras^G12D or Tet2^−/−Nras^G12D mice (CD45.2⁺ cells) were mixed with CD45.1⁺ WT support BM cells and injected into lethally irradiated recipients (CD45.1). In all time points, Tet2^−/−Nras^G12D recipients showed the highest PB chimerism, suggesting that double-mutant BM HSCs have a higher competitive advantage compared to WT or single-mutant cells (mean chimerism at 16 weeks 92.61 %, p<0.01; WT 41.98 %, Tet2^−/− 86.4 %, Nras^G12D 84.4 %) (Figure S2U). Together, these data suggest that Tet2^−/−Nras^G12D cells can propagate similar CMML-like disease and possess significant competitive advantage in vivo.

Tet2^−/−Nras^G12D Cells Show Activated Ras Signaling and Hypersensitivity to GM-CSF

We next sought to delineate mechanisms of cooperativity, and hypothesized that concurrent Tet2 and Nras mutations would further augment MAPK signaling in vivo. Magnetic isolation of BM or splenic Mac1⁺ cells was performed to assess Ras signaling in phenotypically defined myeloid population and confirmed high purity of Mac1 positive isolation (Figure S3A). Western blot (WB) analysis showed higher levels of pErk1/2 and pAkt in Tet2^−/−Nras^G12D Mac1⁺ myeloid cells compared to WT or single-mutant Mac1⁺ cells (Figure 3A). Whole splenocytes from leukemic Tet2^−/−Nras^G12D mice also exhibited higher levels of pRaf1, pMek, pErk1/2, pAkt and pS6 compared to WT or single-mutant cells, although cellular heterogeneity in whole splenocytes could underlie some of the differences (Figure S3B). Phospho-flow analysis confirmed higher expression of pErk, pAkt and pS6 in whole splenic cells and Mac1⁺ myeloid cells derived from Tet2^−/−Nras^G12D mice (Figure 3B and S3C). In immunofluorescence (IF) examination, Mac1⁺ cells in nodular lesions were strongly positive for pErk1/2 in the liver of Tet2^−/−Nras^G12D mice, whereas most Mac1⁺ cells were negative for pErk1/2 in WT or single-mutant mice (Figure 3C and S3D). These data indicate that Tet2 loss and Nras mutation synergistically activate Ras signaling much greater than either allele alone. Consistent with these data, we observed greater accumulation of active Ras-GTP in both Tet2^−/−Nras^G12D Mac1⁺ cells and whole splenocytes compared to WT or single-mutant cells (Figure S3E–S3H).

Figure 3. Tet2 Loss and Nras Mutation Synergistically Activate Ras Signaling and Sensitize Cells to GM-CSF.

(A) WB analysis for pErk1/2, Erk1/2, pAkt, Akt and Vinculin in BM or splenic Mac1⁺ myeloid cells derived from WT (BM), Tet2^−/− (spleen), Nras^G12D (spleen) and Tet2^−/−Nras^G12D (spleen) mice at 6 months from PIPC injection.

(B) Histogram of phospho-flow analysis for pErk, pAkt and pS6 in whole splenic cells and Mac1⁺ splenic cells derived from WT, Nras^G12D and leukemic Tet2^−/−Nras^G12D mice at 6 months from PIPC injection.

(C) Representative images of immunofluorescence for pErk1/2 and Mac1 in livers derived from WT, Tet2^−/−, Nras^G12D and Tet2^−/−Nras^G12D mice at 7 months from PIPC injection. Green cells indicate pErk1/2 positive cells and red cells indicate Mac1 positive cells. The scale bars represent 100 μm.

(D) WB analysis for pErk1/2, Erk1/2, pAkt, Akt and Vinculin in BM or splenic Mac1⁺ cells stimulated with or without GM-CSF.

(E) BM cells from WT, Nras^G12D and Tet2^−/−Nras^G12D mice (3 months from PIPC injection) were plated in cytokine free methylcellulose media with a range of GM-CSF concentrations. The graph shows the colony number ratio in each cytokine concentration relative to GM-CSF 10 ng/mL arm (n=3 for each group). *; p=0.0124 (Nras^G12D vs Tet2^−/−Nras^G12D), p=8.9253×10⁻⁵ (WT vs Tet2^−/−Nras^G12D), **; p=9.7880×10⁻⁵ (Nras^G12D vs Tet2^−/−Nras^G12D), p=0.0016 (WT vs Tet2^−/−Nras^G12D), ***; p=8.1217×10⁻⁶ (Nras^G12D vs Tet2^−/−Nras^G12D), p=0.0006 (WT vs Tet2^−/−Nras^G12D).

An unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S3.

Monocytosis and hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) are hallmarks of CMML (Padron et al., 2013). Given the increase in MAPK activation, we hypothesized that Tet2^−/−Nras^G12D cells would show a synergistic cytokine hypersensitivity. Tet2^−/−Nras^G12D Mac1⁺ cells stimulated with GM-CSF demonstrated higher levels of pErk1/2 and pAkt compared to cytokine-stimulated WT or single-mutant Mac1⁺ cells (Figure 3D), consistent with augmentation of signaling by GM-CSF. This was also the case in GM-CSF stimulated double-mutant whole splenocytes, albeit the existence of cellular heterogeneity in this population could contribute (Figure S3I). Moreover, Tet2^−/−Nras^G12D cells generated a higher number of colonies in GM-CSF supplemented methylcellulose media compared to WT or Nras^G12D cells (Figure 3E and S3J). Of note, Tet2^−/−Nras^G12D cells did not show hypersensitivity to interleukin-3 (IL-3), whose receptor shares a major signaling subunit of GM-CSF receptor (Hercus et al., 2009) (Figure S3K). These data suggest that Tet2^−/−Nras^G12D cells show selective hypersensitivity to GM-CSF leading to activated signaling and cell proliferative output, consistent with features of human CMML.

Concurrent Tet2 and Nras^G12D Mutations Cooperatively Suppress Negative Regulators of Ras Signaling

We next sought to elucidate the potential mechanism by which concurrent Tet2 loss augments MAPK signaling in Nras^G12D mutant leukemia cells. Given the mechanistic link between Tet2 loss-of-function and epigenetic silencing, we hypothesized that Tet2 loss could result in suppression of negative regulators of the MAPK pathway. Known negative regulators of RAS signaling include neurofibromin (NF1), which is a GTPase-activating protein (GAP), dual specificity phosphatase (DUSP) family members, which are MAPK phosphatases, and SPROUTY (SPRY) family members, which inhibit RAS or RAF activation (Cichowski et al., 2001; Edwin et al., 2009; Murphy et al., 2010). Tet2^−/−Nras^G12D cells showed decreased mRNA expression of many Dusp/Spry family members compared to WT or Nras^G12D cells (Figure 4A), consistent with broad suppression of a MAPK feedback transcriptional program. In order to assess transcriptional output of these negative regulators in phenotypically defined myeloid population, we checked mRNA expression of these targets in BM or splenic Mac1⁺ myeloid cells. Strikingly, among the MAPK negative effectors, the most significantly reduced in expression in Tet2^−/−Nras^G12D Mac1⁺ cells was Spry2, a known tumor suppressor gene, that is epigenetically silenced in B-cell lymphoma (Frank et al., 2009) (Figure S4A and 4B). This was also observed at the protein level (Figure 4C and S4B). Bisulfite sequencing of the cytosine guanine dinucleotide (CpG) island in the Spry2 promoter demonstrated hypermethylation in Tet2^−/−Nras^G12D cells compared to WT or single-mutant cells and also leukemic Tet2^−/−Flt3^ITD cells (Figure S4C and 4D). Together, these data suggest that concurrent Tet2 and Nras^G12D mutations cooperatively suppress negative regulators of Ras signaling including Spry2.

Figure 4. Tet2 Loss and Nras Mutation Cooperatively Suppress Negative Regulators of Ras Signaling.

(A) Relative mRNA expression of negative regulators of Ras signaling in whole splenic cells derived from WT, Nras^G12D and Tet2^−/−Nras^G12D mice at 6 months from PIPC injection. Results are presented as the ratio of the Nras^G12D or Tet2^−/−Nras^G12D value to the WT value (n=3 for each group). *; p<0.05, **; p<0.01, ***; p<0.001.

(B) Relative mRNA expression of Spry2 in Mac1⁺ BM or splenic cells derived from WT, Tet2^−/−, Nras^G12D and Tet2^−/−Nras^G12D mice at 5 months from PIPC injection. Results are presented as the ratio of the Tet2^−/−, Nras^G12D or Tet2^−/−Nras^G12D value to the WT value (n=3 for each group).

(C) WB analysis for Spry2 and Vinculin in BM or splenic Mac1⁺ cells derived from WT, Tet2^−/−, Nras^G12D and Tet2^−/−Nras^G12D mice at 6 months from PIPC injection.

(D) Bisulfite sequencing of a CpG island located in the mouse Spry2 promoter region.

(E) WB analysis for Spry2 and Vinculin in control (MIGR1) or ectopic Spry2 expressing Tet2^−/−Nras^G12D BM cells.

(F) Serial replating assay of control (MIGR1) or ectopic Spry2 expressing Tet2^−/−Nras^G12D BM cells (n=3 for each group).

(G) WB analysis for pRaf1, Raf1, pErk1/2, Erk1/2, pAkt, Akt and Vinculin in control (MIGR1) or ectopic Spry2 expressing Tet2^−/−Nras^G12D BM cells.

(H) Representative immunophenotype of donor derived (GFP⁺) BM Lineage⁻ and LSK fraction of mice transplanted with control (MIGR1) or ectopic Spry2 expressing Tet2^−/−Nras^G12D BM cells. BM cells were collected from Tet2^−/−Nras^G12D mice at 6 months from PIPC injection and transduced with either GFP-expressing MIGR1 control vector or Spry2 expressing vector. GFP⁺ cells were sort collected and injected to irradiated recipient.

An unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S4.

Restoration of Spry2 Expression Suppresses Signaling and Leukemogenicity of Tet2/Nras Mutant Leukemia Cells

We next assessed whether restoring Spry2 expression would alter HSC self-renewal, signaling, and leukemic engraftment in Tet2^−/−Nras^G12D cells. We expressed Spry2 using a green fluorescent protein (GFP)-expressing vector in Tet2^−/−Nras^G12D BM cells and confirmed stable Spry2 protein expression (Figure 4E). Of note, these cells showed similar Spry2 mRNA expression level as WT BM cells, providing rationale for this system (Figure S4D). Tet2^−/−Nras^G12D cells expressing control GFP showed serial replating capacity, whereas cells expressing Spry2 lost this capacity, consistent with disrupted HSC self-renewal (Figure 4F). By contrast, expression of Spry2 in Tet2^−/− or Nras^G12D cells did not alter colony number or serial replating capacity (Figure S4E). WB analysis revealed decreased expression of pRaf1, pErk1/2, and pAkt in Tet2^−/−Nras^G12D cells expressing Spry2 compared to controls (Figure 4G). Phospho-flow analysis further confirmed reduced Erk phosphorylation in Spry2 expressing Mac1⁺ cells (Figure S4F and S4G). These data clearly show that restoring Spry2 expression attenuates Ras signaling output. We next transplanted Tet2^−/−Nras^G12D BM cells expressing Spry2 or control GFP into recipient mice. Mice engrafted with Tet2^−/−Nras^G12D GFP cells showed engraftment of donor-derived leukemic cells in lineage⁻ and LSK compartments (BM and spleen); by contrast recipient mice transplanted with Tet2^−/−Nras^G12D Spry2 cells showed near complete loss of engraftment of leukemic cells in HSPCs compartments (Figure 4H and S4H). To determine if restoration of Spry2 expression affects BM homing, we injected Tet2^−/−Nras^G12D GFP cells or Tet2^−/−Nras^G12D Spry2 cells into irradiated mice (Figure S4I). Tet2^−/−Nras^G12D Spry2 cells showed attenuated homing capacity compared to control cells (Figure S4J), suggesting that Spry2 silencing is crucial for both homing and reconstitution of Tet2^−/−Nras^G12D leukemic cells.

To further clarify the functional role of Spry2 in Tet2^−/−Nras^G12D cells, we knocked down Spry2 in Nras^G12D cells by transducing Nras^G12D BM cells with either control or 2 independent Spry2 targeting GFP-labeled shRNA vectors and assessed signaling activation as well as replating capacity. In both shSpry2 cases, GFP⁺ Nras^G12D cells showed higher levels of pErk1/2 and pAkt compared to control cells (Figure S4K–S4L), consistent with augmented Ras signaling upon Spry2 knockdown. Importantly, both Spry2-knockdown Nras^G12D cells enhanced replating capacity in vitro (Figure S4M), suggesting that Spry2 silencing contributes to HSC self-renewal in Tet2^−/−Nras^G12D cells. Collectively, these data validate Spry2 as a key functional epigenetic target in Tet2^−/−Nras^G12D cells.

Tet2^−/−Nras^G12D Driven Leukemia is Sensitive to MEK Inhibition

Preclinical studies have demonstrated efficacy with MEK inhibition in Nras^G12D driven AML (Burgess et al., 2014). Binimetinib, a potent MEK inhibitor, showed significant activity in patients with NRAS-mutated advanced melanoma (Ascierto et al., 2013) but has not been investigated in NRAS-mutant hematologic malignancies. Given Tet2^−/−Nras^G12D cells show highly activated MAPK signaling, we hypothesized that binimetinib would show increased efficacy in Tet2^−/−Nras^G12D leukemia. WB analysis showed significantly decreased pErk1/2 in binimetinib treated cells compared to vehicle, consistent with on-target pathway inhibition (Figure 5A). We next explored the efficacy of binimetinib in vivo in mice transplanted with Tet2^−/−Nras^G12D cells using a previously described protocol (Burgess et al., 2014) (Figure S5A). Binimetinib treatment significantly improved overall survival of Tet2^−/−Nras^G12D leukemia mice compared to vehicle treatment (median survival 6.0 weeks versus 2.7 weeks vehicle, p<0.0001) (Figure 5B). Tet2^−/−Nras^G12D leukemia mice were also treated with either vehicle or binimetinib for 24 days after engraftment; in this context MEK inhibitor therapy also improved survival in drug treated mice (Figure S5B and S5C). Binimetinib treatment improved splenomegaly (mean 112 mg, p=0.0147; vehicle 316 mg) (Figure 5C, S5D, and S5E) and reversed the myeloid cell infiltration in target organs (Figure 5D). Most importantly, binimetinib therapy significantly reduced CD45.2⁺ leukemic burden in both BM and spleen (Figure S5F and 5E). We also assessed the sensitivity of Tet2^−/−Nras^G12D leukemia cells to 2 other MEK inhibitors, selumetinib and PD-0325901 (PD-032) (Burgess et al., 2014; Jänne et al., 2013). Ex vivo treatment of Tet2^−/−Nras^G12D cells with either selumetinib (1 μM) or PD-032 (1 μM) significantly decreased pErk1/2 in these cells compared to vehicle (Figure S5G). We next treated Tet2^−/−Nras^G12D transplant leukemia mice with either vehicle or selumetinib in vivo (Figure S5H and S5I). Selumetinib treatment significantly improved overall survival compared to vehicle treatment (median survival 6.1 weeks versus 4.0 weeks vehicle, p<0.0001) (Figure S5J). It also significantly decreased WBC and monocyte count in PB (Figure S5K), improved hepatomegaly (Figure S5L) and reduced CD45.2⁺ leukemic burden in PB, BM and spleen of Tet2^−/−Nras^G12D leukemia mice (Figure S5M). In vivo PD-032 treatment significantly reduced donor-derived Mac1⁺ myeloid cells in spleen of Tet2^−/−Nras^G12D leukemia mice as well (Figure S5N and S5O). These data demonstrate that MEK inhibition by multiple MEK inhibitors shows robust preclinical efficacy in Tet2^−/−Nras^G12D leukemia.

Figure 5. Tet2^−/−Nras^G12D Driven Leukemia Model is Sensitive to MEK Inhibition.

(A) WB analysis for pErk1/2, Erk1/2 and Vinculin in Tet2^−/−Nras^G12D splenic cells ex vivo treated with either vehicle or binimetinib.

(B) Kaplan Meier survival curve of Tet2^−/−Nras^G12D driven leukemia model upon treatment with either vehicle or binimetinib 20 mg/kg BID.

(C) Representative images of spleens at 17 days of treatment with vehicle or binimetinib 20 mg/kg BID. Spleens from age matched control mice are also shown (n=3 for each group).

(D) Representative images of HE staining of liver, spleen and lung at 17 days of treatment with vehicle or binimetinib 20 mg/kg BID. The scale bars represent 200 μm.

(E) Leukemic burden as reflected by CD45.2⁺ donor chimerism in BM and spleen after 11 days of treatment with vehicle or binimetinib 20 mg/kg BID (n=3 for each group).

(F) Peripheral WBC counts, hematocrit percentage, platelet counts, neutrophil percentage, monocyte percentage and lymphocyte percentage of secondary recipients transplanted with vehicle or binimetinib treated Tet2^−/−Nras^G12D cells 3 weeks after transplantation (n=7 for each group).

(G) The percentage of CD45.2⁺ donor chimerism and representative immunophenotype of CD45.2⁺ donor cell fraction in PB derived from secondary recipients transplanted with vehicle or binimetinib treated Tet2^−/−Nras^G12D cells 3 weeks after transplantation (n=7 for each group).

(H) Kaplan Meier survival curve of secondary recipients transplanted with vehicle or binimetinib treated Tet2^−/−Nras^G12D cells.

A log-rank test was used for survival statistics. Otherwise, an unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S5.

We next treated mice engrafted with CD45.2 Tet2^−/−Nras^G12D leukemic cells followed by serial transplantation and checked leukemia development in secondary recipients (Figure S5P). CBC of secondary recipients at 3 weeks from transplantation revealed a significant improvement in anemia, thrombocytopenia and monocytosis in recipients engrafted with MEK inhibitor treated cells compared to vehicle cell recipients (Figure 5F). Furthermore, mice engrafted with binimetinib treated Tet2^−/−Nras^G12D cells showed a significantly lower leukemic burden as reflected by CD45.2⁺ donor cell chimerism in PB compared to mice engrafted with vehicle treated cells (Figure 5G). Binimetinib therapy in primary recipients led to significant improvement in the survival of secondary recipients, demonstrating binimetinib treatment can diminish the leukemia-initiating capacity of Tet2^−/−Nras^G12D cells in vivo (Figure 5H).

Given Spry2 silencing by promoter hypermethylation may lead to enhanced MAPK signaling and activity of DNA hypomethylating agents (HMA) in Tet2^−/−Flt3^ITD AML model (Shih et al., 2017), we assessed the efficacy of HMA in Tet2^−/−Nras^G12D driven leukemia. 5-Azacytidine (5-Aza) treatment did not show survival benefit in Tet2^−/−Nras^G12D leukemia mice (Figure S5Q and S5R). Unlike binimetinib, 5-Aza treatment led to persistent splenomegaly and elevated pErk1/2 in whole splenocytes (Figure S5S–S5U). In vivo 5-Aza treatment neither restored Spry2 expression nor removed Spry2 promoter hypermethylation in Tet2^−/−Nras^G12D cells (Figure S5V and S5W). These data suggest that Tet2^−/−Nras^G12D driven leukemia is resistant to 5-Aza therapy and that HMA therapy does not restore Spry2 expression.

Preferential Sensitivity of TET2/NRAS Double-Mutant Leukemia to MEK Inhibition

Although previous studies have suggested that NRAS-mutant leukemias might be sensitive to MEK inhibition, most NRAS-mutant leukemia patients do not respond to MAPK targeted therapy (Borthakur et al., 2016), suggesting there are additional biological covariates that modulate the response to MEK inhibition. Given the efficacy of MEK inhibition in our model, we asked if the presence of concurrent alterations in Tet2 sensitizes Nras mutant leukemic cells to MEK inhibition. In vitro, Tet2^−/−Nras^G12D Mac1⁺ myeloid cells derived from mice 6 months from PIPC injection with a diagnosis of CMML were more sensitive to MEK inhibition, with a lower half maximal inhibitory concentration (IC₅₀) to binimetinib compared to Nras^G12D Mac1⁺ cells in a cell viability assay (IC₅₀, Tet2^−/−Nras^G12D 70.91 nm versus Nras^G12D 1472 nm, p<0.0001) (Figure 6A). Similarly, Tet2^−/−Nras^G12D whole splenocytes and BM cells also showed preferential sensitivity to binimetinib compared to Nras^G12D cells (Figure S6A and S6B).

Figure 6. Preferential Sensitivity of TET2/NRAS Double-Mutant Leukemia to MEK Inhibition.

(A) Sensitivity of Nras^G12D and Tet2^−/−Nras^G12D splenic Mac1⁺ cells to binimetinib after 48 hr of drug exposure relative to untreated control. IC₅₀ of each arm are also shown. Data are representative of three independent experiments.

(B) Experimental outline for comparing the sensitivity of Nras^G12D and Tet2^−/−Nras^G12D driven leukemia to binimetinib in vivo.

(C) Spleen weights at 12 days of treatment with vehicle or binimetinib 20 mg/kg BID (n=5 for each group).

(D) Leukemic burden as reflected by CD45.2⁺ donor chimerism and representative immunophenotype of CD45.2⁺ donor cell fraction in BM after 12 days of treatment with vehicle or binimetinib 20 mg/kg BID (n=5 for each group).

(E) WB analysis for pRAF1, RAF1, pERK1/2, ERK1/2, pAKT, AKT, pS6, S6 and VINCULIN in NRAS^G12D mutant human AML cell line THP-1 cells with or without TET2 knockdown.

(F) Growth curve of THP-1 cells with or without TET2 knockdown treated with vehicle (DMSO) or binimetinib 0.1 μM in vitro (n=3 for each group).

(G) Ratio of GFP⁺ cells relative to day1 in THP-1 cells with or without TET2 knockdown treated with vehicle (DMSO) or binimetinib 0.1 μM in vitro (n=3 for each group).

(H) CFU assay cultured with human GM-CSF (10 ng/mL) and various concentration of binimetinib using healthy cord blood-MNCs and BM-MNCs derived from NRAS^G12D mutant myelodysplastic syndromes (MDS) patient or TET2^H702fsNRAS^G12S double mutant AML patient (patient #1) (data derived from cells plated in triplicate).

An unpaired Student’s t test was used for p values. Data shown in graphs indicate mean ± S.D. See also Figure S6.

Binimetinib therapy reversed splenomegaly and reduced BM leukemic burden in Tet2^−/−Nras^G12D mice, whereas MEK inhibition did not attenuate spleen size or BM leukemic burden in Nras^G12D mice (Figure 6B–6D). Similar results were seen in leukemic cell chimerism in BM Mac1⁺ and lineage⁻ fractions and in the percentage of donor-derived BM and splenic Mac1⁺ and lineage⁻ compartments (Figure S6C and S6D). We observed similar selectivity for MEK inhibition in Tet2^−/−Nras^G12D leukemia cells compared to Nras^G12D alone when employing a more aggressive transplant model without wild type support cells (Figure S6E–S6J and 6B–6D). We next treated WT and Tet2^−/−Nras^G12D cell recipients with either vehicle or binimetinib (20 mg/kg, bis in die (BID)) for 11 days followed by CBC analysis. Binimetinib therapy caused no apparent change in CBCs of WT recipients, whereas it significantly improved thrombocytopenia and monocytosis in Tet2^−/−Nras^G12D recipients (Figure S6K), indicating that binimetinib is specifically active in Tet2^−/−Nras^G12D leukemia without impacting normal hematopoiesis. Taken together, these data demonstrate Tet2^−/−Nras^G12D leukemic cells are more sensitive to MEK inhibition than Nras^G12D or wild type cells.

To validate these findings in a human leukemia context, we knocked down TET2 using GFP-labeled shRNA vectors in NRAS^G12D mutant THP-1 cells, a human AML cell line (Janssen et al., 1987) (Figure S6L). Liquid culture of TET2-knockdown THP-1 cells with GM-CSF led to skewed differentiation toward CD14⁺CD15⁻ monocytic lineage at the expense of CD14⁻CD15⁺ granulocytic compartment (Figure S6M), consistent with previous studies of TET2 knockdown in human cord blood (CB) cells (Pronier et al., 2011). WB analysis showed increased levels of pRAF1, pERK1/2, pAKT and pS6 in TET2-knockdown THP-1 cells compared to control cells, consistent with increased RAS signaling output upon TET2 inactivation with NRAS mutation (Figure 6E). In proliferation assays, MEK inhibition abrogated TET2-knockdown THP-1 cell growth more potently than control cell growth, suggesting TET2 knockdown in THP-1 cells further sensitizes to MEK inhibition (Figure 6F). Due to the relative drug resistance of a small fraction of non-knockdown GFP negative cells, GFP percentage progressively decreased in TET2-knockdown THP-1 cells upon binimetinib exposure, whereas it remained stable in control cells, consistent with selective activity of MEK inhibition on the TET2-knockdown THP-1 clone (Figure 6G). TET2-knockdown THP-1 cells showed higher level of pERK1/2, pAKT and pS6 upon GM-CSF stimulation (Figure S6N) and generated a higher number of colonies in GM-CSF supplemented methylcellulose media compared to control cells (Figure S6O), indicating similar hypersensitivity to GM-CSF in TET2-knockdown THP-1 cells. We next assessed the impact of TET2 knockdown on NRAS^Q61L mutant OCI-AML3 cells and FLT3-ITD positive MOLM13 cells (Nonami et al., 2015; Quentmeier et al., 2003). Hairpin-transduced OCI-AML3 and MOLM13 cells demonstrated efficient knockdown of TET2 (Figure S6P and S6Q). Liquid culture of TET2-knockdown NRAS-mutant OCI-AML3 cells led to similar skewed differentiation toward CD14⁺CD15⁻ monocytic lineage at the expense of CD14⁻CD15⁺ granulocytic compartment (Figure S6R). Conversely, liquid culture of TET2-knockdown FLT3-ITD positive MOLM13 cells led to the opposite immunophenotypic differentiation pattern (Figure S6S). MEK inhibition similarly abrogated cell growth and dramatically decreased GFP positivity in TET2-knockdown OCI-AML3 cells compared to control cells but not in TET2-knockdown MOLM13 cells (Figure S6T and S6U). These data clearly suggest that TET2 is a key biological covariate that regulates both myelomonocytic differentiation and sensitivity to MEK inhibition selectively in NRAS-mutant human AML but not in FLT3-ITD positive human AML.

We next performed apoptosis and cell cycle analysis (Figure S6V). Binimetinib exposure led to an increased proportion of apoptotic cells in TET2-knockdown THP-1 cells compared to control cells (Figure S6W). In addition, binimetinib treatment caused shift toward G₀+G₁ phase at the expense of proliferative cell state (S/G₂/M phase) in TET2-knockdown THP-1 cells (Figure S6X). These data indicate that MEK inhibition induces both apoptosis and decreased proliferation in TET2-knockdown THP-1 cells.

To assess the drug sensitivity in primary patient samples, we collected healthy donor CB mononuclear cells (MNC) and BMMNC from NRAS^G12D mutant and TET2^H702fsNRAS^G12S double-mutant patient samples. Consistent with murine and human cell line data, MEK inhibition with binimetinib significantly inhibited colony formation of TET2^H702fsNRAS^G12S double-mutant cells more potently than healthy control or NRAS^G12D mutant cells (Figure 6H). We confirmed higher sensitivity to MEK inhibitor in TET2/NRAS double-mutant cells derived from 3 additional independent patients compared to healthy control and NRAS single-mutant cells (Figure S6Y). Overall, these data suggest that TET2/NRAS double-mutant primary patient samples show increased sensitivity to MEK inhibition.

DISCUSSION

Mutations in epigenetic modifiers and signaling factors frequently co-occur in myeloid malignancies (Papaemmanuil et al., 2016; Patel et al., 2012). These studies led us to hypothesize that TET2 and NRAS mutation functionally cooperate to promote myeloid transformation. We show that concurrent Tet2/Nras mutations cooperatively lead to transcriptional silencing of negative regulators of the MAPK pathway, leading to synergistic activation of Ras signaling. Consistent with these observations, Tet2/Nras mutant leukemia shows increased sensitivity to MEK inhibition, suggesting that TET2 mutations represent a biomarker that predicts for response to MAPK targeted therapies. We show that concurrent Tet2^−/− and Nras^G12D alterations in hematopoietic cells cooperate in vivo, leading to lethal CMML with enhanced self-renewal compared to mice expressing either mutant disease allele alone. These data contrast with ectopic expression studies which did not show cooperativity (Chang et al., 2014), and demonstrate the importance of using tissue-specific conditional alleles to investigate mutational cooperativity and stem cell function in vivo. Moribund Tet2^−/−Nras^G12D mice showed monocytosis, and marked extramedullary myeloid infiltration to a greater extent than mice expressing either allele alone, suggesting that multiple organ failure caused by robust myeloid infiltration accounts for the demise in Tet2^−/−Nras^G12D mice. It is noteworthy that mice transplanted with Tet2^−/−Nras^G12D cells also developed a similar leukemic phenotype, consistent with a cell intrinsic effect of these alleles in leukemogenesis.

We also demonstrated that concurrent Tet2 and Nras^G12D mutations lead to augmentation of Ras signaling. Previous studies in murine leukemia models and in epithelial tumor systems have shown that mutant Ras expression is not sufficient to hyperactivate MAPK signaling or induce overt transformation, and retroviral insertion mutagenesis studies have shown that additional genetic events which augment MAPK signaling can induce leukemic transformation and MEK dependence (Haigis et al., 2008; Lauchle et al., 2009). However, these studies did not identify specific somatic human disease alleles that can serve to augment MAPK signaling. Here we show that mutations in a common co-occurring disease allele, TET2, can serve as an additional genetic event that attenuates MAPK feedback and increases MAPK signaling. Recent work demonstrated that Trp53 loss combined with Nras^G12D causes highly penetrant AML in vivo with hyperactivation of MAPK signaling in myeloid progenitors which was mediated by increased expression of oncogenic Nras (Zhang et al., 2017). Allelic imbalance favoring mutant Kras expression can also lead to increased MEK sensitivity (Burgess et al., 2017). By contrast, we found that Spry2 and genes encoding other MAPK negative feedback effectors are transcriptionally silenced in Tet2^−/−Nras^G12D leukemia cells, thereby elucidating a mechanism of oncogenic cooperativity between an epigenetic mutation and a signaling effector. Homing and reconstitution assay revealed that Spry2 silencing is crucial for leukemia initiation in Tet2^−/−Nras^G12D driven leukemia.

Our studies suggest that TET2 mutations, when co-occurring with RAS mutations, induce increased sensitivity to MEK inhibition, which may identify a subset of patients at highest likelihood for response to targeted MAPK inhibition. In addition, MEK inhibition can diminish leukemia-initiating capacity of Tet2^−/−Nras^G12D cells in vivo, consistent with a dependence on MAPK signaling in Tet2^−/−Nras^G12D leukemia stem cells. Although several studies have reported functional cooperativity of mutations in epigenetic modifiers and signaling factors (Abdel-Wahab et al., 2012; Shih et al., 2015), the impact of co-occurring disease alleles on the sensitivity to targeted therapy has not been well delineated. We demonstrate in murine models, human cell culture systems and primary patient samples that Tet2^−/−Nras^G12D leukemia cells are more sensitive to MEK inhibition compared to Nras^G12D cells. Notably, we have shown in human AML cells that loss of TET2 selectively cooperates with mutant NRAS alleles but not with FLT3-ITD to modulate myeloid differentiation and response to MEK inhibition. The preferential sensitivity of TET2/NRAS double-mutant leukemia to MEK inhibition may be due to induction of both apoptosis and decreased proliferation in these cells upon drug treatment, whereas MEK inhibitors induce mainly cell cycle arrest in Nras-mutant AML (Burgess et al., 2014). Our data also inform a potential role for MEK inhibitor therapy in NRAS mutant leukemia patients with concurrent mutations in TET2, IDH1/2 or WT1 that should be investigated in the clinical context; this is further underscored by a recent report of an exceptional response to MEK inhibition in a patient with TET2/NRAS co-mutant leukemia (Khanna et al., 2015).

In contrast to MEK inhibition, 5-Aza treatment did not improve survival of Tet2^−/−Nras^G12D leukemia mice. This was further corroborated by the findings that 5-Aza therapy neither restored Spry2 expression nor removed Spry2 promoter hypermethylation in Tet2^−/−Nras^G12D cells, leading to persistent MAPK signaling. These data indicate that Tet2^−/−Nras^G12D leukemia is resistant to HMA therapy, justifying inhibition of MAPK signaling by MEK inhibitor rather than restoration of Spry2 expression by HMA as a potential therapeutic strategy for TET2/NRAS mutant leukemia. Our group recently reported that Tet2^−/−Flt3^ITD AML model is highly sensitive to 5-Aza, whereas IDH2-mutant AML patients with concurrent NRAS or other MAPK pathway mutations are resistant to IDH2 inhibition (Amatangelo et al., 2017; Shih et al., 2017). Collectively, these studies suggest that concurrent mutations in signaling factors modulate clinical efficacy of HMA in TET2 mutant leukemia and that NRAS mutations may drive resistance to specific epigenetic therapies, which require future mechanistic investigation.

Taken together, our data suggest a mechanism of cooperativity between mutations in epigenetic regulators and Ras pathway mutations, and inform a model of Tet2/Nras mutant leukemogenesis (Figure 7). In this model, Tet2 mutations promote clonal expansion and “epigenetic heterogeneity” such that different loci are epigenetically altered in different populations, including a subset of cells with silencing of MAPK negative regulators. If this clone of cells subsequently acquires a somatic activating Nras mutation, this leads to hyperactivated Ras signaling and clonal expansion, which results in leukemia progression. However, as these clones are highly dependent on activated Ras signaling, they are preferentially sensitive to MEK inhibition (Figure 7). Our data indicates that cooperative epigenetic remodeling by TET2 loss and NRAS mutation drives myeloid transformation and MEK inhibitor sensitivity, which provides a rationale for mechanism based therapy. Our studies suggest that further investigations of the mechanisms of cooperativity between co-occurring oncogenic disease alleles can elucidate dependencies with therapeutic relevance.

Figure 7. Proposed Model of Tet2/Nras Mutant Leukemogenesis.

The first step is acquisition of Tet2 mutation in HSPCs. This causes clonal expansion and epigenetic heterogeneity in Tet2-mutated clones, where promoters of multiple genes become hyper-methylated including that of Spry2. Once Tet2-mutated/Spry2 promoter hyper-methylated clone acquires additional Nras mutation, this causes robust activation of Ras signaling and clonal selection/expansion of this specific clone, which leads to leukemia progression. However, as these clones are highly dependent on Ras signaling, they are highly sensitive to MEK inhibition.

STAR METHODS

Detailed methods are provided in the online version of this paper and include the following:

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
FITC anti-mouse CD11b	BioLegend	Cat#101205; RRID: AB_312788
PE anti-mouse CD11b	BioLegend	Cat#101207; RRID: AB_312790
APC/Cy7 anti-mouse CD11b	BioLegend	Cat#101225; RRID: AB_830641
PE anti-mouse Gr-1	BioLegend	Cat#108407; RRID: AB_313372
APC/Cy7 anti-mouse Gr-1	BioLegend	Cat#108423; RRID: AB_2137486
PE anti-mouse CD71	BioLegend	Cat#113807; RRID: AB_313568
APC anti-mouse Ter119	BioLegend	Cat#116211; RRID: AB_313712
APC/Cy7 anti-mouse Ter119	BioLegend	Cat#116223; RRID: AB_2137788
APC/Cy7 anti-mouse NK-1.1	BioLegend	Cat#108723; RRID: AB_830870
APC/Cy7 anti-mouse B220	BioLegend	Cat#103223; RRID: AB_313006
APC/Cy7 anti-mouse CD3	BioLegend	Cat#100221; RRID: AB_2057374
APC/Cy7 anti-mouse CD19	BioLegend	Cat#115529; RRID: AB_830706
APC/Cy7 anti-mouse CD4	BioLegend	Cat#100413; RRID: AB_312698
PE anti-mouse CD117/cKit	BioLegend	Cat#105807; RRID: AB_313216
APC anti-mouse CD117/cKit	BioLegend	Cat#105811; RRID: AB_313220
PE/Cy7 anti-mouse Sca-1	BioLegend	Cat#108113; RRID: AB_493597
APC anti-mouse Sca-1	BioLegend	Cat#108111; RRID: AB_313348
PE anti-mouse CD150	eBioscience	Cat#12-1502-82; RRID: AB_1548765
PerCP/Cy5.5 anti-mouse CD48	BioLegend	Cat#103421; RRID: AB_1575045
eFluor 450 anti-mouse CD16/32	eBioscience	Cat#48-0161-82; RRID: AB_1272191
eFluor 450 anti-mouse CD45.1	eBioscience	Cat#48-0453-82; RRID: AB_1272189
PE/Cy7 anti-mouse CD45.2	BioLegend	Cat#109829; RRID: AB_1186103
APC/Cy7 anti-mouse CD45.2	BioLegend	Cat#109823; RRID: AB_830788
PerCP/Cy5.5 anti-human CD14	eBioscience	Cat#45-0149-42; RRID: AB_1518736
APC anti-human CD14	eBioscience	Cat#17-0149-42; RRID: AB_10669167
PE anti-human CD15	BD Biosciences	Cat#555402; RRID: AB_395802
Rabbit anti-mouse Ki-67	Abcam	Cat#ab16667; RRID: AB_302459
Rabbit anti-mouse/human pRaf1	Cell Signaling	Cat#9427S; RRID: AB_2067317
Rabbit anti-mouse/human Raf1	Cell Signaling	Cat#9422S; RRID: AB_390808
Rabbit anti-mouse/human pMek	Cell Signaling	Cat#9121S; RRID: AB_331648
Rabbit anti-mouse/human Mek	Cell Signaling	Cat#8727S; RRID: AB_10829473
Rabbit anti-mouse/human pErk1/2	Cell Signaling	Cat#4370S; RRID: AB_2315112
Rabbit anti-mouse/human Erk1/2	Cell Signaling	Cat#9102S; RRID: AB_330744
Rabbit anti-mouse/human pAkt	Cell Signaling	Cat#4060S; RRID: AB_2315049
Rabbit anti-mouse/human Akt	Cell Signaling	Cat#4691S; RRID: AB_915783
Rabbit anti-mouse/human pS6	Cell Signaling	Cat#2211S; RRID: AB_331679
Rabbit anti-mouse/human S6	Cell Signaling	Cat#2217S; RRID: AB_331355
Rabbit anti-mouse/human Spry2	Cell Signaling	Cat#13264
Rabbit anti-mouse/human Vinculin	Cell Signaling	Cat#4650S; RRID: AB_10559207
Allophycocyanin AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch	Cat#711-136-152; RRID: AB_2340601
Rabbit anti-mouse CD11b (Mac1)	Abcam	Cat#ab133357; RRID: AB_2650514
Biotinylated goat anti-rabbit IgG	Vector Laboratories	Cat#PK-6101; RRID: AB_2336820
Goat anti-rabbit IgG and Alexa Fluor 488 Tyramide	Invitrogen	Cat#T20922
Goat anti-mouse IgG and Alexa Fluor 568 Tyramide	Invitrogen	Cat#T20914
Bacterial and Virus Strains
Biological Samples
Human CB MNC	New York Blood Bank	N/A
Human BM MNC	Hospital for Special Surgery	N/A
Patient samples	Memorial Sloan	Table S1
	Kettering Cancer Center
Chemicals, Peptides, and Recombinant Proteins
Binimetinib	ChemieTek	Cat#CT-A162
Selumetinib	ChemieTek	Cat#CT-A6244
PD-0325901	ChemieTek	Cat#CT-PD03
5-Azacytidine	Sigma	Cat#A2385
Carboxymethylcellulose sodium salt	Sigma	Cat#C4888
(2-hydroxypropyl)-β-cyclodextrin	Sigma	Cat#H107
(Hydroxypropyl) methyl cellulose	Sigma	Cat#09963
Tween 80	Fisher	Cat#BP338
Polyinosinic:polycytidylic acid	Amersham	Cat#27-4732-01
Bovine serum albumin	Fisher	Cat#BP1600-1
RPMI medium	MSKCC Media Core	N/A
RPMI, 2mM L-glutamine	MSKCC Media Core	N/A
Fetal bovine serum	MSKCC Media Core	N/A
Fetal calf serum	MSKCC Media Core	N/A
Penicillin-Streptomycin	Fisher	Cat#15140122
Ammonium chlorider	Sigma	Cat#A9434
Potassium bicarbonate	Fisher	Cat#P184-500
MethoCult GF M3434	StemCell Technologies	Cat#03434
MethoCult M3231	StemCell Technologies	Cat#03231
MethoCult H4230	StemCell Technologies	Cat#04230
Recombinant mouse GM-CSF	Peprotech	Cat#315-03
Recombinant mouse IL-3	Peprotech	Cat#213-13
Recombinant mouse SCF	Peprotech	Cat#250-03
Recombinant human GM-CSF	Peprotech	Cat#300-03
Recombinant human IL-6	Peprotech	Cat#200-06
Dimethyl sulfoxide	Fisher	Cat#D128-500
16% Paraformaldehyde aqueous solution	Electron Microscopy Sciences	Cat#15710
Decalcifier I	Leica Biosystems	Cat#3800440
Epitope Retrieval Solution 2 BOND	Leica Biosystems	Cat#AR9640
3,3’-Diaminobenzidine tetrahydrochloride hydrate	Sigma	Cat#32750
EZ Prep (10X)	Ventana Medical Systems	Cat#950-102
Cell Conditioning 1 (CC1)	Ventana Medical Systems	Cat#950-124
Background Buster	Innovex Biosciences	Cat#NB306
DAPI	Sigma	Cat#D9542
RNase-free DNase set (50)	Qiagen	Cat#79254
VeriQuest Fast SYBR Green qPCR Master Mix	Affymetrix	Cat#75675
Polybrene	American Bio	Cat#AB01643
HEPES buffer	Fisher	Cat#BP299
Critical Commercial Assays
Fixation/Permeabilization Solution Kit	BD Biosciences	Cat#554714
PE mouse anti-Ki-67 Set	BD Biosciences	Cat#556027; RRID: AB_2266296
FIX & PERM Cell Permeabilization Kit	Invitrogen	Cat#GAS004
PE Annexin V Apoptosis Detection Kit I	BD Biosciences	Cat#559763
EasySep Mouse PE Positive Selection Kit	StemCell Technologies	Cat#18554
Active Ras Pull-Down and Detection Kit	Fisher	Cat#16117
Bond Polymer Refine Detection Kit	Leica Biosystems	Cat#DS9800
DISCOVERY DABMap Detection Kit	Ventana Medical Systems	Cat#760-124
RNeasy Mini Kit (50)	Qiagen	Cat#74104
RNeasy Micro Kit (50)	Qiagen	Cat#74004
Verso cDNA Synthesis Kit	Fisher	Cat#AB1453A
AllPrep DNA/RNA Micro Kit	Qiagen	Cat#80284
EZ DNA Methylation Kit	Zymo Research	Cat#D5001
TOPO TA Cloning Kit for Sequencing	Invitrogen	Cat#450071
CellTiter-Glo Luminescent Cell Viability Assay	Promega	Cat#G7571
Deposited Data
Experimental Models: Cell Lines
THP-1	ATCC	Cat#TIB-202; RRID: CVCL_0006
OCI-AML3	DSMZ	Cat#ACC-582; RRID: CVCL_1844
MOLM13	DSMZ	Cat#ACC-554; RRID: CVCL_2119
293T	ATCC	Cat#CRL-3216; RRID: CVCL_0063
Experimental Models: Organisms/Strains
Mouse: Mx1-Cre Tet2f/f	Moran-Crusio et al., 2011	N/A
Mouse: NrasG12D	The Jackson Laboratory	Cat#008304: RRID: IMSR_JAX:008304
Mouse: Mx1-Cre	The Jackson Laboratory	Cat#003556: RRID: IMSR_JAX:003556
Mouse: Mx1-Cre Tet2f/f Flt3ITD	Shih et al., 2015	N/A
C57BL/6J	The Jackson Laboratory	Cat#000664: RRID: IMSR_JAX:000664
B6.SJL-Ptprca/BoyAiTac	Taconic	Cat#4007: RRID: IMSR_TAC:4007
Oligonucleotides
Quantitative RT-PCR: primer sequences	See Table S2	N/A
Bisulfite sequencing: mouse Spry2 F 5’-GTTGGCGAGTTTTTTTTTTTTTTGG-3’	Integrated DNA Technologies	N/A
Bisulfite sequencing: mouse Spry2 R 5’-CGCCTACCTCCACTTAAAAACAAC-3’	Integrated DNA Technologies	N/A
Short hairpin sequence: mouse Spry2 hairpin#1 5’-TAATATTTTGTGACTGTGCCAT-3’	MSKCC RNAi Core	N/A
Short hairpin sequence: mouse Spry2 hairpin#2 5’-TTTATATTGAACATAGCATCTG-3’	MSKCC RNAi Core	N/A
Short hairpin sequence: human Scramble 5’-GCCGGCAGCTAGCGACGCCAT-3’	Pronier et al., 2011	N/A
Short hairpin sequence: human TET2 5’-GGGTAAGCCAAGAAAGAAA-3’	Pronier et al., 2011	N/A
Recombinant DNA
pCR4-TOPO	Invitrogen	Cat#450071
3ME6/mSprouty2	Addgene	Cat#22097
Gateway pENTR1A Dual Selection Vector	Invitrogen	Cat#A10462
pMSCV-IRES-GFP	Shih et al., 2015	N/A
pMSCV-miRE-LEPG	MSKCC RNAi Core	N/A
EcoPack plasmids	Shih et al., 2015	N/A
pRRLsin-PGK-eGFP-WPRE	Pronier et al., 2011	N/A
PsPAX2	Pronier et al., 2011	N/A
pMD2.G	Pronier et al., 2011	N/A
Software and Algorithms
Prism 6	GraphPad	https://www.graphpad.com
ImageJ version 2.0.0	N/A	https://imagej.nih.gov/ij/
FlowJo V9.9.6	Tree Star	https://www.flowjo.com/
Other

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ross L. Levine (leviner@mskcc.org).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Transgenic Animals

All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at Memorial Sloan Kettering Cancer Center. The conditional Mx1-Cre⁺;Tet2^f/f mice, Mx1-Cre⁺;Nras^G12D mice and Mx1-Cre⁺;Tet2^f/f;Flt3^ITD mice are all C57BL/6 background and are previously described (Li et al., 2011; Moran-Crusio et al., 2011; Shih et al., 2015). All WT mice used in the experiments are Mx1-Cre⁺ and injected with PIPC. 4–6 week-old Mx1-Cre⁺;WT, Mx1-Cre⁺;Tet2^f/f, Mx1-Cre⁺;Nras^G12D and Mx1-Cre⁺;Tet2^f/f;Nras^G12D mice were injected with PIPC to induce gene deletion/recombination. Deletion/recombination was confirmed 2 weeks after PIPC injection and mice were used for experiments at least 3 months after PIPC injection. Males and females were used for experiments. Recipients were C57BL/6J or B6.SJL-Ptprc^a/BoyAiTac female mice between 6–12 weeks of age. C57BL/6J and B6.SJL-Ptprc^a/BoyAiTac mice were purchased from The Jackson Laboratory and Taconic, respectively.

Cell Lines

293T cells were cultured in DME with 10%FCS and penicillin/streptomycin. THP-1 cells (human acute monocytic leukemia cell line originated from 1 year-old male patient) were cultured in RPMI with 10%FCS, 2mM L-glutamine and penicillin/streptomycin. OCI-AML3 (human acute myeloid leukemia cell line originated from 57 year-old male patient) and MOLM13 cells (human acute myeloid leukemia cell line originated from 20 year-old male patient) were cultured in RPMI with 10%FCS and penicillin/streptomycin. All cell line cultures were performed in 37°C/5% CO₂ condition. THP-1 and MOLM13 cells were authenticated by short tandem repeat profiling. OCI-AML3 was recently procured from a cell repository, therefore testing was not performed.

Patient Samples

Human BM samples were obtained from de-identified patients at Memorial Sloan Kettering Cancer Center. Patient characteristics are depicted in Table S1. To establish controls, CB samples were obtained from normal full-term deliveries after signed informed consent from New York Blood Bank. Normal healthy BM sample was obtained from a de-identified orthopedic patient without an evidence of hematologic malignancies at Hospital for Special Surgery. All patients provided informed consent. Approval was obtained from the Institutional Review Board at Memorial Sloan Kettering Cancer Center and Hospital for Special Surgery, and conducted in accordance to the Declaration of Helsinki protocol.

METHOD DETAILS

Peripheral Blood Analysis

Blood was collected by retroorbital bleeding using heparinized microhematocrit capillary tubes (Thermo Fisher Scientific). Automated peripheral blood counts were obtained using a ProCyte Dx (IDEXX Laboratories) according to the manufacturer’s protocol.

Flow Cytometry and Fluorescence-Activated Cell Sorting

For surface flow cytometry and cell sorting of mouse PB, BM, and spleen, red blood cells (RBCs) were lysed and stained with monoclonal antibodies in PBS plus 1 % BSA for 1 hr on ice. For flow cytometry of erythroid lineage, BM or splenic cells were stained without RBC lysis. Intracytoplasmic staining was performed with the Fixation/Permeabilization Solution kit from BD Biosciences. Cell cycle analysis was performed with the PE Mouse Anti-Ki-67 Set (BD Biosciences) and the FIX & PERM Cell Permeabilization Kit (Invitrogen). Apoptosis analysis was performed with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). DAPI was used for both cell cycle and apoptosis analysis. See Supplemental Information for antibodies. Cell populations were analyzed using an LSR Fortessa (Becton Dickinson) and sorted with a FACSAria II instrument (Becton Dickinson). Data were analyzed with FlowJo software (Tree Star).

Antibodies

Antibodies used for flow cytometry were as follows: (anti-mouse) CD11b (M1/70), Gr1 (RB6-8C5), CD71 (R17217), Ter119 (TER-119), NK1.1 (PK136), CD45R (RA3-6B2), CD3 (17A2), CD19 (6D5), CD4 (GK1.5), cKit (2B8), Sca1 (D7), CD150 (mShad150), CD48 (HM48-1), CD16/32 (93), CD45.1 (A20), and CD45.2 (104). All anti-mouse antibodies were purchased from BioLegend, eBioscience or BD Biosciences. The “lineage cocktail” included NK1.1, CD11b, CD45R, CD3, Gr1, Ter119, CD19, and CD4. (anti-human) CD14 (61D3, eBioscience) and CD15 (HI98, BioLegend). Antibodies used for phospho-flow analysis were as follows: (primary antibodies) Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (Cell Signaling), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling), Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (Cell Signaling). (secondary antibody) Allophycocyanin (APC) AffiniPure F(ab’)₂ Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories)

Bone Marrow Transplantation

Dissected femurs and tibias were isolated. Bone marrow was flushed with a syringe into RPMI 10 % fetal calf serum (FCS) media. Spleens were isolated and single cell suspensions were made by mechanical disruption using glass slides. RBCs were lysed in ammonium chloride-potassium bicarbonate lysis buffer for 10 min on ice. For serial transplantation of splenic cells, 2×10⁶ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 1×10⁶ competitor BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated (2x550 Rad) CD45.1 host mice in primary transplantation. 2×10⁶ whole splenic cells taken from the first recipient mice 9 weeks after injection mixed with 1×10⁶ competitor BM cells (CD45.1) were transplanted into lethally irradiated CD45.1 host mice in secondary transplantation. For transplantation to check disease transplantability to sublethally irradiated mice, 1×10⁶ whole donor splenic cells (CD45.2, 1 year from PIPC injection) without support BM cells were transplanted via tail vein injection into sublethally irradiated (600 rads) CD45.2 host mice. For transplantation to check survival of sublethally irradiated mice, 1×10⁶ whole donor splenic cells (CD45.2, 1 year from PIPC injection) without support BM cells were transplanted via tail vein injection into sublethally irradiated (450 rads) CD45.1 host mice. For competitive repopulation assay, 5×10³ sorted BM LSK cells (CD45.2) mixed with 2×10⁵ competitor BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For transplantation to check leukemic engraftment of Spry2 restored BM cells, GFP⁺ cells were sort collected and 1×10⁵ GFP⁺ cells (CD45.2) mixed with 1×10⁵ support BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For formal homing assays, GFP⁺ cells were sort collected and 2×10⁵ GFP⁺ cells (CD45.2) without support BM cells were transplanted via tail vein injection into lethally irradiated CD45.1 host mice, followed by sacrifice 15 hr after injection. For binimetinib drug trial to check survival (Figure S5A and 5B), 1×10⁶ whole donor splenic cells (CD45.2, 1 year from PIPC injection) without support BM cells were transplanted via tail vein injection into sublethally irradiated (600 rads) CD45.2 host mice. For binimetinib drug trial to check survival, splenomegaly and pathology (Figure 5C–5D and S5B–S5E), 5×10³ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 2×10⁵ support BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For binimetinib drug trial to check leukemic burden, hematologic toxicity, and sensitivity to binimetinib (1^st trial in which treatment was initiated before leukemic engraftment, Figure 6B–6D, S6C–S6D and S6K), 2×10⁶ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 2×10⁵ support BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For 2^nd drug trial to check sensitivity to binimetinib in more established disease model (2^nd trial in which treatment was initiated after leukemic engraftment, Figure S6E–S6J), 2×10⁵ whole donor splenic cells (CD45.2, 1 year from PIPC injection) without support BM cells were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For selumetinib and PD-0325901 drug trial to check survival, CBCs and leukemic burden, 5×10⁴ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 2×10⁵ support BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For secondary transplantation to check leukemia-initiating capacity, 5×10³ whole splenic cells taken from the first recipient mice 2 weeks after injection mixed with 2×10⁵ support BM cells (CD45.1) were transplanted into lethally irradiated CD45.1 host mice. For 5-AZA drug trial to check survival, 5×10³ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 5×10⁵ support BM cells (CD45.1) were transplanted via tail vein injection into lethally irradiated CD45.1 host mice. For binimetinib and 5-AZA drug trial to check splenomegaly, signaling and Spry2 expression/methylation, 5×10³ whole donor splenic cells (CD45.2, 1 year from PIPC injection) mixed with 2×10⁵ support BM cells (CD45.2) were transplanted via tail vein injection into lethally irradiated CD45.2 host mice.

Isolation of Mac1⁺ cells

Isolation of Mac1⁺ cells was performed with EasySep Mouse PE Positive Selection Kit (18554, STEMCELL Technologies), EasySep Magnet (18000, STEMCELL Technologies) and PE-conjugated anti-mouse CD11b (M1/70).

In Vitro Colony Forming Assays

For serial replating assay, sorted GFP⁺ BM cells were seeded in triplicate into cytokine supplemented methylcellulose medium (Methocult, M3434, STEMCELL Technologies). Colonies propagated in culture were counted at day 7. Cells were resuspended and re-plated at 10,000 cells per well. For cytokine sensitivity assay using Tet2^−/−Nras^G12D cells, BM cells from WT, Nras^G12D and Tet2^−/−Nras^G12D mice (3 months from PIPC injection) were plated at 10,000 cells per well in cytokine free methylcellulose medium (Methocult, M3231, STEMCELL Technologies) with various concentrations of recombinant mouse GM-CSF or recombinant mouse IL-3 (0 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL). For cytokine sensitivity assay using control or TET2-knockdown THP-1 cells, sorted GFP⁺ THP-1 cells were plated at 10,000 cells per well in cytokine free methylcellulose medium (Methocult, H4230, STEMCELL Technologies) with various concentrations of recombinant human GM-CSF (0 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL). Colonies propagated in culture were counted at day 7. For colony forming assay using primary human patient samples, human CB-MNCs and BM-MNCs were thawed and were seeded in 6 well plates at 4.0×10⁴, 2.0×10⁵ or 1.0×10⁶ cells per well density in triplicate into cytokine free methylcellulose medium (Methocult, H4230, STEMCELL Technologies) with GM-CSF 10 ng/mL and various concentrations of binimetinib (Vehicle, 0.1 μM and 1 μM). Plates were placed into an incubator at 37 °C and 5 % CO₂ for 14 days and colony counts were determined. Binimetinib was suspended in dimethyl sulfoxide (DMSO). DMSO was used as a vehicle.

Western Blot Analysis

The following antibodies were used for western blot analysis: pRaf1 (Cell Signaling), Raf1 (Cell Signaling), pMek (Cell Signaling), Mek (Cell Signaling), pErk (Cell Signaling), Erk (Cell Signaling), pAkt (Cell Signaling), Akt (Cell Signaling), pS6 (Cell Signaling), S6 (Cell Signaling), Spry2 (Cell Signaling), and Vinculin (Cell Signaling). Active Ras pull-down assay was performed with the Active Ras Pull-Down and Detection Kit (Thermo Scientific). For GM-CSF stimulation assay, whole splenic cells and isolated BM/splenic Mac1⁺ cells collected from WT, Tet2^−/−, Nras^G12D and Tet2^−/−Nras^G12D mice at 3 months from PIPC injection or sort collected GFP⁺ control or TET2-knockdown THP-1 cells were stimulated with or without GM-CSF (10 ng/mL) for 15 min in serum free media. Protein was extracted for western blot analysis following cytokine stimulation. For checking Spry2 expression in Spry2 transduced cells, BM cells were collected from Tet2^−/−Nras^G12D mice at 5 months from PIPC injection and transduced with either GFP-expressing MIGR1 control vector or Spry2 expressing vector. GFP⁺ cells were sort collected and protein was extracted for western blot analysis. For western blot analysis for pErk in ex vivo treated cells, whole splenic cells were collected from leukemic mice transplanted with Tet2^−/−Nras^G12D splenic cells and ex vivo treated with either vehicle, binimetinib (5 μM), selumetinib (1 μM) or PD-0325901 (1 μM) for 1 hr. Protein was extracted after treatment for analysis. For TET2-knockdown THP-1 cells, THP-1 cells were transduced with either GFP-expressing Scr control vector or shTET2 expressing vector. GFP⁺ cells were sort collected and protein was extracted for analysis.

Pathology

Dissected tissue samples were fixed in 4 % paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Bone samples were decalcified with Decalcifier I (Leica) for 24 hr, followed by washing with sterilized water for 30 min and embedded in paraffin. Paraffin blocks were sectioned at 4 μm and stained with H&E or reticulin. Ki-67 immunohistochemistry staining was performed on a Leica Bond™ RX using the Bond™ Polymer Refine Detection Kit (Cat. No. DS9800). Sections were stained with Ki-67 (Abcam, Cat. Ab16667, diluted at 1:100), were pre-treated using heat mediated antigen retrieval with EDTA pH9 (Leica Biosystem Epitope Retrieval Solution 2, Cat. No. AR9640) for 20 min. Diaminobenzidine was used as the chromogen, counterstained with hematoxylin and mounted.

Immunofluorescence

The immunofluorescent staining was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking (Ventana Medical Systems) for 8 min. Slides were incubated with anti-pErk1/2 (Cell Signaling, cat# 4370, 1 μg/ml) and anti-Mac1 (abcam, cat#ab133357, 1 μg/ml) for 5 hr, followed by 60 min incubation with biotinylated goat anti-rabbit (Vector Labs, cat# PK6101, 5.75 μg/ml) at 1:200 dilution. The detection was performed with Streptavidin-HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen, cat# T20922, for pErk1/2) and Tyramide Alexa Fluor 568 (Invitrogen, cat#T20914, for Mac1) prepared according to the manufacturer’s protocol with predetermined dilutions. After staining slides were counterstained with DAPI (Sigma Aldrich, cat# D9542, 5 μg/ml) for 10 min and coverslipped with Mowiol.

Quantitative RT-PCR

Total RNA was isolated from whole BM cells, whole splenic cells, isolated Mac1⁺ cells, sorted GFP⁺ BM cells or sorted GFP⁺ THP-1/OCI-AML3/MOLM13 cells using RNeasy Mini/Micro Kit (Qiagen) according to the manufacturer’s protocol. RNA was treated with RNase-free DNase set (Qiagen) to remove contaminating genomic DNA. cDNA was synthesized using Verso cDNA Synthesis Kit (Thermo Fisher Scientific). The quantity of cDNA was normalized according to the expression of mouse Gapdh, mouse Actb, or human HPRT measured by real-time RT-PCR using VeriQuest Fast SYBR Green qPCR Master Mix (Affymetrix) and QuantStudio 7 Flex System (Applied Biosystems). Data were analyzed by the delta Ct ratio technique using housekeeping genes. The sequences of the primers used for the amplification of each gene are listed in Table S2.

Bisulfite Sequencing

DNA was extracted from whole splenic cells derived from WT, Tet2^−/−, Nras^G12D, Tet2^−/−Nras^G12D and Tet2^−/−Flt3^ITD mice at 6 months from PIPC injection or whole splenic cells from Tet2^−/−Nras^G12D transplant leukemia mice treated with vehicle or 5-Aza 5 mg/kg IP using All Prep micro kit (Qiagen). DNA was subjected to bisulfite conversion with the EZ DNA Methylation Kit (Zymo Research) and PCR amplified. The primers used for the amplification of bisulfite converted DNA are as follows: mouse Spry2 (Forward primer 5’-GTTGGCGAGTTTTTTTTTTTTTTGG-3’, Reverse primer 5’-CGCCTACCTCCACTTAAAAACAAC-3’). PCR amplified products were subcloned into pCR4-TOPO vector with the TOPO TA Cloning Kit for Sequencing (Invitrogen) and sequenced by Sanger method.

Vector Construction and Viral Transduction

The mouse Spry2 cDNA construct (3ME6/mSprouty2, Addgene Plasmid#22097) was first cloned into the shuttle vector (pENTR1A Dual Selection Vector, Invitrogen) according to the manufacturer’s protocol. Shuttle vector with mouse Spry2 cDNA construct was cloned into MSCV-IRES-GFP vector with the Gateway LR recombination reaction (Invitrogen). Short hairpin construct against mouse Spry2 (hairpin #1; 5’-TAATATTTTGTGACTGTGCCAT-3’, hairpin #2; 5’-TTTATATTGAACATAGCATCTG-3’) cloned into retroviral vector pMSCV.miRE.LEPG and retroviral empty control vector (pMSCV.miRE.LEPG) were provided by RNAi core facility (Memorial Sloan Kettering Cancer Center). Virus was produced by transfecting 293T cells with MSCV and EcoPack plasmids. After RBC lysis, BM cells were cultured in media containing RPMI/10 % fetal bovine serum (FBS) and IL-3 (7 ng/mL), IL-6 (10 ng/mL), and stem cell factor (10 ng/mL). Cells were infected twice in the presence of polybrene and HEPES buffer. Short hairpin construct against human TET2 (5’-GGGTAAGCCAAGAAAGAAA-3’) or Scramble (5’-GCCGGCAGCTAGCGACGCCAT-3’) cloned into lentiviral vector pRRLsin-PGK-eGFP-WPRE were transfected into 293T cells with the packaging vector PsPAX2 and envelope vector pMD2.G to produce virus. THP-1 cells were cultured in media containing RPMI/10 %FBS, 2 mM L-glutamine. To generate stable cell lines, THP-1 cells were infected twice with concentrated lentiviral supernatant in the presence of polybrene and HEPES buffer. THP-1 cells were then expanded and GFP⁺ cells were sort collected by flow cytometry.

Cell Viability Assay

Isolated splenic Mac1⁺ cells or whole BM/splenic cells are derived from Nras^G12D and Tet2^−/−Nras^G12D mice at 6 months from PIPC injection. Cells were plated in 96 well plates at 3×10⁴ cells per well density and exposed to binimetinib in triplicate at various concentrations (0 nM, 1 nM, 2 nM, 10 nM, 20 nM, 100 nM, 200 nM, 1,000 nM, 2,000 nM, 5,000 nM, 10⁴ nM, and 10⁵ nM). Relative cell numbers (compared to vehicle treated control) were estimated after 48 or 72 hr by measuring ATP levels by CellTiter Glo (Promega) for suspension cultures according to the manufacturer’s protocol. DMSO was used as a vehicle.

Human Leukemia Cell Line Experiments

For flow cytometric analysis of human granulomonocytic lineage marker (CD14/CD15) in THP-1 cells, control or TET2-knockdown (sort collected GFP⁺) THP-1 cells were cultured in RPMI/10 %FBS, 2 mM L-glutamine for 3 weeks, followed by stimulation with various concentration of GM-CSF for 5 days. Cells were stimulated in GM-CSF 0.01 ng/mL, 0.1 ng/mL, 1 ng/mL and 10 ng/mL. For flow cytometric analysis of human granulomonocytic lineage marker (CD14/CD15) in OCI-AML3 and MOLM13 cells, control or TET2-knockdown (sort collected GFP⁺) OCI-AML3 or MOLM13 cells were cultured in RPMI/10 %FBS for 3 weeks followed by flow cytometric analysis. For growth curve and GFP positivity analysis, control or TET2-knockdown (sort collected GFP⁺) THP-1, OCI-AML3 or MOLM13 cells were cultured in media (RPMI/10 %FBS, 2 mM L-glutamine for THP-1 cells, RPMI/10 %FBS for OCI-AML3 and MOLM13 cells) for 3 weeks. Cells were then initially plated in 6 well plates at 1.0×10⁶ cells per well density and exposed to vehicle (DMSO) or binimetinib 0.1 μM in triplicate. Total cell numbers and GFP positivity were measured everyday for 1 week using Vi-CELL Cell Counter & Cell Viability Analyzers (Beckman Coulter) or flow cytometry, respectively. For cell cycle and apoptosis analysis, control or TET2-knockdown (sort collected GFP⁺) THP-1 cells were cultured in RPMI/10 %FBS, 2 mM L-glutamine for 3 weeks, followed by exposure to vehicle (DMSO) or binimetinib 0.1 μM for 48 or 72 hr and analyzed.

In Vivo Treatment Studies

For binimetinib study, mice were treated twice a day (BID) using oral gavage with either vehicle (1% carboxymethyl cellulose (CMC) in water) or binimetinib at 20 mg/kg. Binimetinib was suspended in 1 % CMC. For selumetinib and PD-0325901 study, mice were treated once a day (QD) using oral gavage with either vehicle (50 mM 2-hydroxypropyl β cyclodextrin in water for selumetinib study or 5 % hydroxypropyl methylcellulose/2 % Tween80 in water for PD-0325901), selumetinib at 25mg/kg or PD-0325901 at 5 mg/kg. Selumetinib was suspended in 50 mM 2-hydroxypropyl β cyclodextrin and PD-0325901 was suspended in 5 % hydroxypropyl methylcellulose/2 % Tween80. Binimetinib (CT-A162), selumetinib (CT-A6244) and PD-0325901 (CT-PD03) were all purchased from ChemieTek. For 5-Aza study, mice were treated once a day using intraperitoneal injection (IP) with either vehicle (PBS) or 5-Aza at 5 mg/kg. 5-Aza was suspended in PBS and was purchased from SIGMA-ALDRICH (A2385). See Supplemental Information for details of treatment schedule.

Experimental Design

No specific methods were used for randomization and investigators were not blinded to the identity of samples. No statistical methods were utilized to determine sample size. The experiments described in this study were designed to use the minimum number of animals required. Each experiment was performed at least in triplicate to ensure reproducibility.

QUANTIFICATION AND STATISTICAL ANALYSIS

Blood count analyses and organ weights of mice were recorded from at least 3 mice per genotype as indicated in figure legends. Flow cytometric analyses of primary mice were recorded from at least 3 mice per group. Phospho-flow analyses of primary mice for pErk, pAkt and pS6 were performed with a total of 3 mice per genotype. In vitro colony forming assays using murine BM cells and human leukemia cell lines were performed in triplicate in three independent experiments. In vitro colony forming assays using primary patient samples were performed in triplicate with three independent NRAS-mutant and four independent TET2/NRAS double-mutant samples. Active Ras pull-down assays were performed in three independent experiments. For serial transplantation and competitive transplantation experiments, at least 5 recipient mice were used for each donor. Transplantation experiments for in vivo treatment studies with binimetinib, selumetinib, PD-0325901 and 5-Aza were performed with at least 3 recipients per treatment group. Quantitative RT-PCR analyses for Dusp/Spry family members using murine BM/spleen samples were recorded from at least 3 mice per genotype/group. Quantitative RT-PCR analyses for human TET2 in human leukemia cell lines were recorded from 3 independent scrambled or TET2-knockdown cells. In bisulfite sequencing, PCR amplified products were subcloned into pCR4-TOPO vector and at least 10 clones were sequenced for each group. Cell viability assays were performed in three independent experiments. Differentiation, cell cycle, apoptosis, growth curve and GFP positivity analyses in human leukemia cell lines were recorded from 3 independent scrambled or TET2-knockdown cells. The number of animals, cells and experimental replication can be found in the respective figure legend. The unpaired Student’s t test was used to compare the mean of two groups. Data were analyzed and plotted using GraphPad Prism 6 software and Microsoft Excel. Data shown in graphs indicate mean ± S.D. Kaplan-Meier survival analysis and log-rank test were used to compare survival outcomes.

Significance.

Specific combinations of co-occuring disease alleles in myeloid malignancies can cooperatively drive malignant transformation and impact therapeutic response. However, in most cases, the mechanism of cooperativity between concurrent mutations in myeloid transformation with therapeutic relevance is elusive. Here we show that TET2 and NRAS mutations cooperate to induce myeloid leukemia using a mouse model. Tet2 loss and Nras mutation synergistically activate Ras signaling much greater than either allele alone through cooperative suppression of negative regulators of Ras signaling. This leads to an increased dependence on activated Ras signaling and preferential sensitivity to MEK inhibition in TET2/NRAS double-mutant leukemia. Our studies suggest that investigations of the mechanisms of cooperativity between co-occurring oncogenic disease alleles can elucidate dependencies with therapeutic relevance.

Highlights.

• Tet2^−/− and Nras^G12D alleles cooperate to induce myeloid transformation

• Tet2 loss and Nras mutation synergistically activate Ras signaling

• Tet2^−/− and Nras^G12D cooperatively suppress Dusp/Spry regulators including Spry2

• TET2/NRAS co-mutant leukemia is highly sensitive to MEK inhibition