Alisa Damnernsawad

Combined MEK and JAK inhibition abrogates murine myeloproliferative neoplasm

Overactive RAS signaling is prevalent in juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) in humans, and both are refractory to conventional chemotherapy. Conditional activation of a constitutively active oncogenic Nras (NrasG12D/G12D) in murine hematopoietic cells promotes an acute myeloproliferative neoplasm (MPN) that recapitulates many features of JMML and MP-CMML. We found that NrasG12D/G12D-expressing HSCs, which serve as JMML/MP-CMML-initiating cells, show strong hyperactivation of ERK1/2, promoting hyperproliferation and depletion of HSCs and expansion of downstream progenitors. Inhibition of the MEK pathway alone prolonged the presence of NrasG12D/G12D-expressing HSCs but failed to restore their proper function. Consequently, approximately 60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks, and the remaining animals continued to display JMML/MP-CMML-like phenotypes. In contrast, combined inhibition of MEK and JAK/STAT signaling, which is commonly hyperactivated in human and mouse CMML, potently inhibited human and mouse CMML cell growth in vitro, rescued mutant NrasG12D/G12D-expressing HSC function in vivo, and promoted long-term survival without evident disease manifestation in NrasG12D/G12D animals. These results provide a strong rationale for further exploration of combined targeting of MEK/ERK and JAK/STAT in treating patients with JMML and MP-CMML.

Nras G12D/+ promotes leukemogenesis by aberrantly regulating hematopoietic stem cell functions

Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras(G12D/+)) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras(G12D/+) in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras(G12D/+) signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras(G12D/+) serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras(G12D/+) HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras(G12D/+) HSCs and prevents the expansion of Nras(G12D/+) HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis.

Loss of wild-type Kras promotes activation of all Ras isoforms in oncogenic Kras-induced leukemogenesis

Despite the well-established role of oncogenic RAS in promoting tumor formation, whether and how wild-type (WT) Ras inhibits tumorigenesis under physiological conditions remains controversial. Here, we show that in a fraction of endogenous oncogenic Kras-induced hematopoietic malignancies, including acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN), WT Kras expression is lost through epigenetic or genetic mechanisms. Using conditional KrasG12D/− mice, we find that WT Kras deficiency promotes oncogenic Kras-induced MPN, but not T-ALL, in a cell-autonomous manner. Loss of WT Kras rescues oncogenic Kras-mediated hematopoietic stem cell depletion and further enhances granulocyte-macrophage colony-stimulating factor signaling in myeloid cells expressing oncogenic Kras. Quantitative signaling studies reveal that oncogenic Kras but not oncogenic Nras leads to cross-activation of WT Ras, whereas loss of WT Kras further promotes the activation of all Ras isoforms. Our results demonstrate the tumor suppressor function of WT Kras in oncogenic Kras-induced leukemogenesis and elucidate its underlying cellular and signaling mechanisms.

Evaluation of allelic strength of human TET2 mutations and cooperation between Tet2 knockdown and oncogenic Nras mutation.

The TET2 (tet methylcytosine dioxygenase 2) gene encodes a methylcytosine dioxygenase that catalyses the hydrolysis of 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC) and promotes DNA demethylation through passive and active mechanisms (Shih, et al 2012). Loss-of-function mutations in TET2 are identified in patients with myeloid and lymphoid malignancies, and are particularly frequent in patients with chronic myelomonocytic leukaemia (CMML) (36–58%) (Shih, et al 2012). Consistent with the patient sequencing analysis, conditional knockout of Tet2 in mice dysregulates haematopoietic stem cell (HSC) function and promotes development of a myeloid malignancy closely resembling human CMML (Cimmino, et al 2011). Despite the high mutation frequency, the prognostic importance of TET2 mutations is unclear in many cases (Shih, et al 2012). We postulated that this could be due to the differential allelic strengths of distinct TET2 mutations (e.g. amorphic versus hypomorphic) and/or the influence of other concurrent genetic alterations. Although nonsense and frameshift mutations are found spread over the entire TET2 sequence, the majority of missense mutations occur in the two conserved regions of TET2 protein (Figure S1): a cysteine-rich region within 1134-1444 amino acids and a catalytic domain (double strand β helix, DSBH) in 1842-1921 amino acids (Fig. 1A). To determine the allelic strengths of distinct TET2 mutations, we characterized five missense mutations prevalent in the COSMIC database in the context of full length human TET2. Two of them (C1193W and R1261G) are located in the cysteine-rich domain and have not been examined before. The other three mutations (I1873T, H1881Q, and R1896S) are located in the DSBH domain and their equivalent mutations were previously evaluated in mouse Tet2 (Ko, et al 2010). Transient expression of full-length wild-type human TET2 in HEK293T cells showed a predominant nuclear localization (~75%) and concomitant detection of 5hmC in the nucleus (Fig. 1B–1D). We observed that in ~25% of TET2-expressing cells, TET2 protein was distributed in both cytoplasm and nucleus and 5hmC staining was diminished (Figure 1B and 1C). These results suggest that intracellular localization of TET2 influences production of 5hmC. In contrast, the mutant proteins containing C1193W, R1261G, I1873T or H1881Q mutations maintained their nuclear localization but 5hmC levels were not detectable, suggesting that these mutations are amorphic. TET2R1896S mutant only showed partial loss of function, suggesting that this mutation is hypomorphic (Fig. 1D). Importantly, our results of human TET2I1873T and TET2R1896S are not consistent with those obtained from equivalent mouse Tet2 mutants, which did not show diminished 5hmC staining (Ko, et al 2010). This could be due to the differences between human TET2 and mouse TET2, emphasizing the importance of validating discoveries from mouse genes in human genes. Nonetheless, our data indicate that leukaemia-associated TET2 mutations lead to complete or partial loss of TET2 function, providing a rational to further stratify leukaemia patients based on their specific TET2 mutations in future prognostic studies. Fig 1 Missense mutations at the Cys-rich and DSBH regions of human TET2 attenuate its catalytic function Recent work identified a significant synergy between loss-of-TET2 and loss-of other epigenetic regulators (Ezh2 (Muto, et al 2013) and Asxl1 (Abdel-Wahab, et al 2013) ) or NOTCH inactivation (loss of Ncstn) (Lobry, et al 2013) in mice. These results suggest that TET2 mutations might indicate a poor prognosis outcome in CMML patients with concurrent EZH2, ASXL1, or NCSTN mutations. However, the prognostic importance of TET2 mutations in patients with other concurrent mutations, for example, RAS signalling pathway mutations, has not been evaluated. Given the high mutation rate of TET2 in CMML patients, we set out to find and characterize additional gene mutations concurrent with TET2 mutations. We performed whole exome sequence analysis of 5 CMML patients with a normal karyotype and at different stages of CMML development, including 2 collected from patients transformed to acute myeloid leukaemia (AML) with antecedent of CMML, 1 with Type II CMML, and 2 with Type I CMML (Fig. 2A). All of them contained TET2 mutations, including 4 frameshift and 11 missense mutations. Among the missense mutations, L1721W and I1762V were reported before (Kohlmann, et al 2011, Nibourel, et al 2010), L1340R was found in the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), while N7S, Q129K, and N196K have not been described but are absent from the SNP database (http://snp-nexus.org/index.html). Detection of more than one TET2 mutation in individual patients suggests the presence of multiple leukaemic clones. The mutation frequency of TET2 in our small cohort is much higher than previously reported (Shih, et al 2012). This could be due to the small sample size and the selection of myeloproliferative variant of CMML (indicated by high white blood cell and monocyte counts) and transformed AML in our study. Fig 2 Tet2 knockdown does not promote NrasG12D/+-induced CMML Consistent with the mouse studies, our sequencing results revealed that all patients carried ASXL1 mutations and two patients carried EZH2 mutations. However, NCSTN mutations were not detected in any of the patients (Fig. 2A). In addition, four patients carried canonical oncogenic mutations in NRAS or KRAS, and one patient carried the KRASV7E mutation, which has not been reported in human cancers. However, V7 codon was recently suggested as a key residual in regulating oncogenic Kras activity (Maurer, et al 2012). Our finding of concurrent TET2 mutations with oncogenic RAS mutations in CMML patients is consistent with our data-mining result of the COSMIC database (Table S1) and other reports (Table S2). To determine whether loss-of-Tet2 co-operates with oncogenic Ras to promote CMML development, we knocked down Tet2 expression in NrasG12D/+ bone marrow cells (Fig. 2B–2D). Compared with recipients transplanted with control cells expressing a scrambled shRNA, recipients transplanted with control cells expressing Tet2 shRNA (Ko, et al 2010) developed CMML-like phenotypes after a prolonged latency, consistent with previous reports of Tet2 knockout mice (Cimmino, et al 2011). To our surprise, knockdown of Tet2 did not accelerate NrasG12D/+ induced CMML (Fig. 2B) or further promote CMML phenotypes (Fig. 2C and 2D). All CMML mice displayed comparably enlarged spleen and significantly higher percentage of monocytes (Mac1+ Gr1−) and neutrophils (Mac1+ Gr1+) in peripheral blood compared to controls. It is likely that Tet2 knockdown does not result in long-term abrogation of Tet2 expression. Alternatively, oncogenic Ras signalling might alter the subcellular localization of TET2 protein to promote Tet2 loss-of-function during CMML development as shown in BCR-ABL1-driven chronic myeloid leukaemia (Mancini, et al 2012). Thus, further downregulation of Tet2 expression in NrasG12D/+ bone marrow cells does not significantly accelerate CMML progression. It is also possible that the order of mutational acquisition is important. However, current technologies do not allow us to assess this possibility under physiological conditions. In summary, our results provide a rationale to further stratify leukaemia patients based on their specific TET2 mutations and presence of specific additional genetic mutations in future prognostic studies.

Kras is Required for Adult Hematopoiesis

Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its role in adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow (BM) is mediated by Vav‐Cre or inducible Mx1‐Cre. We find that loss of Kras leads to greatly reduced thrombopoietin (TPO) signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while stem cell factor‐evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term‐ and intermediate‐term HSC compartments and a bias toward myeloid differentiation in MPPs. Although granulocyte macrophage colony‐stimulating factor (GM‐CSF)‐evoked ERK1/2 activation is only moderately decreased in Kras‐/‐ myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9‐12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras‐/‐ BM cells is greatly compromised, which is attributable to defects in the self‐renewal of Kras‐/‐ HSCs and defects in differentiated hematopoietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM‐CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. Stem Cells 2016;34:1859–1871

The mystery of oncogenic KRAS: Lessons from studying its wild-type counter part

ABSTRACT Using conditional knock-in mouse models, we and others have shown that despite the very high sequence identity between Nras and Kras proteins, oncogenic Kras displays a much stronger leukemogenic activity than oncogenic Nras in vivo. In this manuscript, we will summarize our recent work of characterizing wild-type Kras function in adult hematopoiesis and in oncogenic Kras-induced leukemogenesis. We attribute the strong leukemogenic activity of oncogenic Kras to 2 unique aspects of Kras signaling. First, Kras is required in mediating cell type- and cytokine-specific ERK1/2 signaling. Second, oncogenic Kras, but not oncogenic Nras, induces hyperactivation of wild-type Ras, which significantly enhances Ras signaling in vivo. We will also discuss a possible mechanism that mediates oncogenic Kras-evoked hyperactivation of wild-type Ras and a potential approach to down-regulate oncogenic Kras signaling.

Abstract B22: Combined MEK and JAK inhibition rescues mutant hematopoietic stem cell function and provides long-term survival in NrasG12D/G12D mice

Overactive Ras signaling is prevalent in human juvenile and chronic myelomonocytic leukemias (JMML/CMML) that are refractory to conventional chemotherapy. Conditional activation of endogenous NrasG12D/G12D in hematopoietic cells of mice leads to an acute myeloproliferative disease (MPN) that recapitulates many features of JMML and myeloproliferative variant of CMML. We found that as JMML/CMML initiating cells, NrasG12D/G12D hematopoietic stem cells (HSCs) show strong ERK1/2 hyperactivation, hyperproliferation, and depletion with concomitant expansion of downstream progenitors. MEK pathway inhibition alone prolongs the presence of mutant HSCs, but fails to restore their proper function. Consequently, ∼60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks and the remaining animals also displayed significant JMML/CMML-like phenotypes. In contrast, simultaneous inhibition of JAK/STAT signaling, a commonly hyperactivated pathway in human and mouse CMML, more potently inhibits human and mouse CMML cell growth in vitro, sustainably rescues mutant HSC function in vivo, and promotes long-term survival without evident disease manifestation in NrasG12D/G12D mice. Our results provide a strong rationale for combined targeting of MEK/ERK and JAK/STAT in treating JMML and CMML patients. Citation Format: Guangyao Kong, Wunderlich Mark, David Yang, Erik A. Ranheim, Ken H. Young, Jinyong Wang, Yuan-I Chang, Juan Du, Yangang Liu, Sin Ruow Tey, Xinmin Zhang, Mark Juckett, Ryan Mattison, Alisa Damnernsawad, Jingfang Zhang, James C. Mulloy, Jing Zhang. Combined MEK and JAK inhibition rescues mutant hematopoietic stem cell function and provides long-term survival in NrasG12D/G12D mice. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B22. doi: 10.1158/1557-3125.RASONC14-B22

Abstract A56: Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis

Although the Ras genes have long been established as proto-oncogenes, substantial evidence indicates that wild-type (WT) Ras genes act as tumor suppressors in a few solid tumor models. However, recent study by Shannon group discovers that loss of WT Nras does not further promote oncogenic Nras-induced leukemias (Xu et al., 2013), raising the possibility that the earlier finding of WT Ras genes as tumor suppressors restricts to specific Ras isoform(s) and/or specific cell types. Here, we show that in mouse cell lines established from oncogenic Kras-induced acute T-cell lymphoblastic leukemia/lymphoma (T-ALL), WT Kras expression is absent or significantly downregulated due to deletion or epigenetic silencing of the locus. Moreover, WT Kras allele is deleted in ∼30% of primary KrasG12D T-ALL samples but not in pre-leukemia specimens. Our results are consistent with a recent study that WT KRAS allele is deleted in early-stage progenitor ALL patients with an oncogenic KRAS mutation (Zhang, et al., 2012), suggesting that loss of WT Kras expression is a pathological mechanism contributing to T-ALL progression. To test our hypothesis, we generated compound mice carrying a conditional KrasG12D allele, a floxed WT Kras allele and the Mx1-Cre allele. In the absence of pIpC induction, the leaky expression of Cre is sufficient to induce both oncogenic KrasG12D activation and WT Kras deletion in ∼50% of HSCs. We find that KrasG12D/- mice have a much shorter life span than KrasG12D/+ mice and display more severe leukemia phenotypes, including splenomegaly, leukocytosis, thrombocytopenia, and increase of monocytic cells in peripheral blood and spleen. Bone marrow transplantation experiment confirms that loss of WT Kras promotes oncogenic Kras-induced leukemias in a cell-autonomous manner. Further studies demonstrate that loss-of-WT Kras attenuates KrasG12D HSC depletion and enhances GM-CSF signaling in KrasG12D hematopoietic stem/progenitor cells (HSPCs). We are continuously working on the underlying mechanisms. Taken together, our study elucidates the cross-talk between WT and oncogenic Kras in leukemogenesis and provides mechanistic insights of the WT Kras function as a tumor suppressor. Citation Format: Yuan-I Chang, Alisa Damnernsawad, Guangyao Kong, Yangang Liu, Qiang Chang, Jing Zhang. Loss of wild-type Kras promotes oncogenic Kras-induced leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A56. doi: 10.1158/1557-3125.RASONC14-A56