Jingfang Zhang

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.

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.

Loss of CD44 attenuates aberrant GM-CSF signaling in Kras G12D hematopoietic progenitor/precursor cells and prolongs the survival of diseased animals

Loss of CD44 attenuates aberrant GM-CSF signaling in Kras G12D hematopoietic progenitor/precursor cells and prolongs the survival of diseased animals

p53−/− synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53-/- mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53-/- bone marrow cells rapidly develop a highly penetrant AML. We find that p53-/- cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53-/- MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53-/- synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Signaling Profiling at the Single‐Cell Level Identifies a Distinct Signaling Signature in Murine Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho‐flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a “HSC phospho‐flow” method to analyze cytokine signaling in murine HSCs at the single‐cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin− cKit+ cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function and granulocyte macrophage‐colony‐stimulating factor (GM‐CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM‐CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF‐evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. Stem Cells2012;30:1447–1514

Deficiency of β Common Receptor Moderately Attenuates the Progression of Myeloproliferative Neoplasm in NrasG12D/+ Mice.

Background: GM-CSF signaling is important in establishing and maintaining juvenile/chronic myelomonocytic leukemia (JMML/CMML). Results: The common β chain of GM-CSF receptor (βc) is dispensable for the function of CMML-initiating cells, but βc−/− prolongs the survival of CMML-bearing mice. Conclusion: βc−/− slows down CMML progression but does not abrogate its initiation. Significance: Inhibiting GM-CSF signaling might alleviate JMML/CMML symptoms but would not eradicate the disease. Activating Ras signaling is a major driver in juvenile and the myeloproliferative variant of chronic myelomonocytic leukemia (JMML/MP-CMML). Numerous studies suggest that GM-CSF signaling plays a central role in establishing and maintaining JMML/MP-CMML phenotypes in human and mouse. However, it remains elusive how GM-CSF signaling impacts on JMML/MP-CMML initiation and progression. Here, we investigate this issue in a well characterized MP-CMML model induced by endogenous NrasG12D/+ mutation. In this model, NrasG12D/+ hematopoietic stem cells (HSCs) are required to initiate and maintain CMML phenotypes and serve as CMML-initiating cells. We show that the common β chain of the GM-CSF receptor (βc) is dispensable for NrasG12D/+ HSC function; loss of βc does not affect the expansion, increased self-renewal, or myeloid differentiation bias in NrasG12D/+ HSCs. Therefore, βc−/− does not abrogate CMML in NrasG12D/+ mice. However, βc deficiency indeed significantly reduces NrasG12D/+-induced splenomegaly and spontaneous colony formation and prolongs the survival of CMML-bearing mice, suggesting that GM-CSF signaling plays an important role in promoting CMML progression. Together, our results suggest that inhibiting GM-CSF signaling in JMML/MP-CMML patients might alleviate disease symptoms but would not eradicate the disease.

Downregulating Notch counteracts Kras G12D -induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

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