Pathogenesis Of Juvenile Myelomonocytic Leukemia Research Articles

Multi-Omics Profiling of JMML HSPCs Reveals Onco-Fetal Reprogramming and Identifies Novel Prognostic Biomarkers and Therapeutic Targets in High-Risk JMML

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm of early childhood, which is characterized by highly heterogenous clinical outcomes ranging from spontaneous resolution to early relapse after hematopoietic stem cell transplantation (HSCT). Established clinical risk factors as well as characteristic mutational patterns do not fully explain this heterogeneity. Recently, we and others identified DNA methylation subgroups in JMML, which were shown to be the only significant factor for predicting overall survival (Schönung et al., Clinical Cancer Research 2021). Together, these findings suggest a functional role for DNA methylation in the molecular pathogenesis of JMML. To elucidate the impact of aberrant DNA methylation on pathogenesis and disease progression, we leveraged a multi-modal molecular analysis approach. We integrated flow cytometry data with single-cell RNA-seq and ultra-low input whole-genome bisulfite sequencing (WGBS) data in order to dissect the hematopoietic system across patients from all epigenetic subgroups (n=8). With this approach, we aimed to identify novel candidate prognostic biomarkers and therapeutic targets for high-risk JMML patients. We observed conservation of epigenetic subgroups already in the hematopoietic stem and progenitor cell (HSPC) compartment, suggesting that differential methylation might be key to understand pathogenesis and course of the disease in JMML. In addition, single-cell transcriptomics revealed disease-specific aberrations to be most pronounced in the HSPC compartment relative to all other hematopoietic cell types. Consequently, we focused our work on JMML HSPCs, which revealed a high degree of immunophenotypic, transcriptomic and methylomic heterogeneity across JMML risk groups. This included the expression of proto-oncogenes belonging to the AP-1 family, CTNNB1 and MECOM (EVI1). Furthermore, we observed divergent lineage priming across JMML subgroups which affected lymphoid, myeloid and erythroid differentiation programs. Remarkably, subgroup-specific aberrations comprised upregulation of fetal developmental programs in HSPCs from high-risk JMML patients. This is of particular interest regarding the cell-of-origin in JMML, since mutations activating the RAS signaling pathway have been identified in newborn blood samples from children who later developed JMML (Behnert et al., Leukemia 2022). Comparing the methylome data of HSPCs from JMML patients with HSPCs from healthy individuals at different developmental stages (fetal, perinatal, juvenile and adult) suggested progressive epigenomic reprogramming of leukemic HSPCs towards high-risk JMML. As a consequence, we observed signatures of both accelerated aging and aberrant activation of onco-fetal programs. This included the downregulation of postnatal HSPC markers such as AVP and vice versa the upregulation of prenatal markers such as HMGA2 in high-risk HSPCs, which was associated with differential methylation of these genes. Moreover, binding motifs of central hematopoietic and developmental transcription factors such as RUNX1, FLI1, CDX4, and several HOX genes were not only enriched in differentially methylated regions but were also found to be differentially methylated themselves. Systematic comparison of JMML HSPCs with their healthy nearest normal counterparts revealed disease-specific expression of several cell surface markers. The expression patterns appeared to be regulated in a subgroup-specific manner, which nominates these factors as novel prognostic biomarkers in JMML. Moreover, we identified surface markers that were not only differentially methylated but also known drug targets in other entities, which is why we selected these genes as high confidence candidates for further functional validation. In a preclinical patient-derived xenograft (PDX) mouse model, targeted treatment with a therapeutic antibody against one of these candidates efficiently depleted high-risk JMML HSPCs and improved survival of JMML-PDX mice. In conclusion, the personalized molecular analysis of JMML HSPCs disentangled disease-specific heterogeneity and provided novel insights into tumorigenesis. Subgroup-specific epigenetic dysregulation included onco-fetal reprogramming of JMML HSPCs and revealed novel prognostic biomarkers and promising novel drug targets specifically for high-risk JMML.

Genomic and Epigenomic Landscape of Juvenile Myelomonocytic Leukemia.

Simple SummaryJuvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by the constitutive activation of the RAS pathway. In spite of the recent progresses in the molecular characterization of JMML, this disease is still a clinical challenge due to its heterogeneity, difficult diagnosis, poor prognosis, and the lack of curative treatment options other than hematopoietic stem cell transplantation (HSCT). In this review, we will provide a detailed overview of the genetic and epigenetic alterations occurring in JMML, and discuss their clinical relevance in terms of disease prognosis and risk of relapse after HSCT. We will also present the most recent advances on novel preclinical and clinical therapeutic approaches directed against JMML molecular targets. Finally, we will outline future research perspectives to further explore the oncogenic mechanism driving JMML leukemogenesis and progression, with special attention to the application of single-cell next-generation sequencing technologies.Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm of early childhood. Most of JMML patients experience an aggressive clinical course of the disease and require hematopoietic stem cell transplantation, which is currently the only curative treatment. JMML is characterized by RAS signaling hyperactivation, which is mainly driven by mutations in one of five genes of the RAS pathway, including PTPN11, KRAS, NRAS, NF1, and CBL. These driving mutations define different disease subtypes with specific clinico-biological features. Secondary mutations affecting other genes inside and outside the RAS pathway contribute to JMML pathogenesis and are associated with a poorer prognosis. In addition to these genetic alterations, JMML commonly presents aberrant epigenetic profiles that strongly correlate with the clinical outcome of the patients. This observation led to the recent publication of an international JMML stratification consensus, which defines three JMML clinical groups based on DNA methylation status. Although the characterization of the genomic and epigenomic landscapes in JMML has significantly contributed to better understand the molecular mechanisms driving the disease, our knowledge on JMML origin, cell identity, and intratumor and interpatient heterogeneity is still scarce. The application of new single-cell sequencing technologies will be critical to address these questions in the future.

Role of lncRNA Morrbid in PTPN11(Shp2)E76K-driven juvenile myelomonocytic leukemia

AbstractMutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, contribute to ∼35% of cases of juvenile myelomonocytic leukemia (JMML). A common clinical picture in children with JMML is that it presents as a constitutive hyperinflammatory syndrome, partially reminiscent of chronic myelomonocytic leukemia in adults. Thus, a component of JMML is associated with a hyperinflammatory state and abundant innate immune cells such as neutrophils and monocytes. Recently, we showed that the evolutionarily conserved mouse lncRNA Morrbid is specifically expressed in myeloid cells and uniquely represses the expression of the proapoptotic gene Bim to regulate the lifespan of myeloid cells. However, its role in JMML has not been investigated. In this study, we characterized the role of Morrbid and its target Bim, which are significantly dysregulated in Shp2E76K/+-bearing myeloid cells, in driving JMML. Loss of Morrbid in a mouse model of JMML driven by the Shp2E76K/+ mutation resulted in a significant correction of myeloid and erythroid cell abnormalities associated with JMML, including overall survival. Consistently, patients with JMML who had PTPN11, KRAS, and NRAS mutations and high expression of MORRBID manifested poor overall survival. Our results suggest that Morrbid contributes to JMML pathogenesis.

Immunophenotypic and Genetic Overlap between JMML and CMML

Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution.Methods:The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel.CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis.Results:We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance.We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining.Conclusions:Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. DisclosuresTasian:Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Acute Myeloid Leukemia (AML) with Somatic Mutations in PTPN11 Is Associated with Treatment Resistance and Poor Overall Survival

Acute Myeloid Leukemia (AML) with Somatic Mutations in PTPN11 Is Associated with Treatment Resistance and Poor Overall Survival

Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia.

The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell-based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias.

Whole‐exome sequencing and microRNA profiling reveal PI3K/AKT pathway’s involvement in juvenile myelomonocytic leukemia

BackgroundClinical studies and genetic analyses have revealed that juvenile myelomonocytic leukemia (JMML) is caused by somatic and/or germline mutations of genes involved in the RAS/MAPK signalling pathway. Given the vastly different clinical prognosis among individual patients that have had this disease, mutations in genes of other pathways may be involved.MethodsIn this study, we conducted whole‐exome and cancer‐panel sequencing analyses on a bone marrow sample from a 2‐year old juvenile myelomonocytic leukemia patient. We also measured the microRNA profile of the same patient’s bone marrow sample and the results were compared with the normal mature monocytic cells from the pooled peripheral blood.ResultsWe identified additional novel mutations in the PI3K/AKT pathway and verified with a cancer panel targeted sequencing. We have confirmed the previously tested PTPN11 gene mutation (exon 3 181G>T) in the same sample and identified new nonsynonymous mutations in NTRK1, HMGA2, MLH3, MYH9 and AKT1 genes. Many of the microRNAs found to be differentially expressed are known to act as oncogenic MicroRNAs (onco‐MicroRNAs or oncomiRs), whose target genes are enriched in the PI3K/AKT signalling pathway.ConclusionsOur study suggests an alternative mechanism for JMML pathogenesis in addition to RAS/MAPK pathway. This discovery may provide new genetic markers for diagnosis and new therapeutic targets for JMML patients in the future.

Yolk sac erythromyeloid progenitors expressing gain of function PTPN11 have functional features of JMML but are not sufficient to cause disease in mice.

Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN). E9.5 YS EMPs from VavCre+;PTPN11D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11E76K ;ROSAYFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN. YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc.

Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency.

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.

CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen.In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML

AbstractAlthough hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage–colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.

Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

Overexpression Of Leukemia Associated CblY371H Mutation In Transgenic Mice Causes Dosage Dependent Embryonic Lethality

Juvenile Myelomonocytic Leukemia (JMML) is a devastating childhood cancer which is rapidly fatal with infiltration of myeloid cells into multiple organs (Hess, Zutter, Castleberry, & Emanuel, 1996). Based on the observation that uniparental disomy was found in the chromosomal region 11q in JMML patient samples, about 15% of patient samples were found to contain a mutation in c-Cbl (Loh et al., 2009). Moreover, mutant Cbl was also found to be a tumor suppressor gene where a germline mutation results in the predisposition for developing JMML (Niemeyer et al., 2010). The c-Cbl gene encodes a multifunctional adaptor protein which contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif which contain E3 ligase activity, and a C-terminal ubiquitin-associated domain. Previous mutations in myeloid malignancies have been described where mutations occur in the RING finger domain or the linker domain (Caligiuri et al., 2007; Dunbar et al., 2008). Interestingly, a hotspot for mutations at residue 371 exists in JMML patients where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H (Loh et al., 2009). This residue belongs in the linker region of the CBL protein, and it was previously observed that Y371 mediates the binding of c-Cbl to the p85 subunit of PI3 kinase (Blaydes et al., 2001). In vitro, CblY371H mutation does indeed destroy its ligase function resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene was silenced (Niemeyer et al., 2010). However, how mutant Cbl gives rise to JMML and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we tested the oncogenicity of the CblY371H mutation in transgenic mice. Of the 6 founder transgenic mice, we chose L5 line for further analysis because it had the highest level of expression. As expected, overexpression of CblY371H by itself in wild type mice had no phenotype since inactivation of wild type Cbl allele is seen in patients. We, then, generated transgenic mice which were heterozygous for both the BAC transgene and the cbl null mutation (CblY371H; Cbl+/-) and then bred them to Cbl heterozygous knockout mice (Cbl+/-). Because homozygous knockout mice have reduced fertility, we preferentially utilized heterozygous mice for the breeding (El Chami et al., 2005). Interestingly, out of the first 118 progeny genotyped where 1/8 of the pups is expected to be the desired genotype (CblY371H;Cbl-/-), no pups were of the desired genotype suggesting embryonic lethality. Because mice which lack both c-cbl and cbl-b are embryonic lethal, we reasoned that high expression that is achieved from the L5 line was causing a dominant negative affect on cbl-b function resulting in an embryonic lethal phenotype. We then tested if a lower level of the transgene expression would result in viable pups. We used the K5 line which was the second highest expressing transgenic line. We were now able to generate the desired genotype (CblY371H; Cbl-/-) although at less than expected numbers. Thus far, we have generated 4 mice which have the correct genotype which are being monitored for disease. We also monitored the heterozygous mice that carried the transgene (CblY371H; Cbl+/-) which were also asymptomatic without any obvious phenotype. Further studies are being done to characterize the hematopoietic phenotype in these mice by isolating fetal liver cells followed by transplantation into lethally irradiated mice and will be discussed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Phosphatidylinositol-3-Kinase p110d Uniquely Promotes Gain-Of-Function PTPN11-Induced GM-CSF Hypersensitivity In a Model Of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a fatal leukemia affecting children under the age of 4 years and is characterized by myelomonocytic cell overproduction and hypersensitivity to GM-CSF. The only curative therapy is allogeneic stem cell transplantation; however, half of children relapse after this aggressive therapy. Approximately 85% of JMML patients bear loss-of-function (LOF) mutations in NF1 or CBL or gain-of-function (GOF) mutations in KRAS, NRAS, or PTPN11. Typically, these mutations are non-overlapping, with the net effect being Ras hyperactivation. Children bearing somatic GOF mutations within PTPN11, which encodes the protein tyrosine phosphatase, Shp2, exhibit the poorest prognosis. GOF Shp2 (Shp2D61Y and Shp2E76K) induces hyperactivation of both the Ras-MEK- Erk and PI3K-Akt pathways. While the Ras-MEK-Erk pathway clearly contributes to the pathogenesis of JMML, we hypothesize that the PI3K-Akt pathway cooperates with the Ras-MEK-Erk pathway to promote JMML. Recently published work indicates that genetic disruption of the PI3K regulatory subunit, p85a, reduces GOF Shp2-induced hypersensitivity to GM-CSF. However, as PI3K regulatory subunits cannot be easily inhibited pharmacologically, we examined the contribution of class IA PI3K catalytic subunits in GOF Shp2-induced JMML.Shp2D61Y/+;Mx1Cre+ mice were crossed with mice bearing conditional knockout of p110a (Pik3caflox/flox) or bearing a kinase dead mutant of p110d (Pik3cdD910A/D910A). Shp2D61Y/+;Mx1Cre-, Shp2D61Y/+;Mx1Cre+, Shp2D61Y/+;Mx1Cre+; Pik3caflox/flox, and Shp2D61Y/+;Mx1Cre+; Pik3cdD910A/D910A mice were treated with polyI;polyC, and 8 weeks post-treatment, animals were euthanized followed by evaluation of spleen size, hypersensitivity of bone marrow low density mononuclear cells (LDMNCs) to GM-CSF, frequency of bone marrow phenotypically-defined common myeloid, granulocyte-monocyte, and megakaryocyte-erythroid progenitors (CMPs, GMPs, and MEPs), and GM-CSF-stimulated Erk and Akt activation. Genetic disruption of p110a failed to normalize GOF Shp2-induced splenomegaly, GM-CSF hypersensitivity in proliferation assays and methylcellulose-based progenitor assays, or hyperphosphorylation of Erk or Akt. In contrast, genetic ablation of p110d kinase activity significantly reduced spleen size, normalized progenitor hypersensitivity to GM-CSF, and reduced both Akt and Erk hyperactivation. Additionally, genetic inhibition of p110d normalized the skewed hematopoietic progenitor distribution reported in the Shp2D61Y/+;Mx1Cre+ mice, while genetic disruption of p110a failed to do so. This unique function of p110d in the context of GOF Shp2-expressing mice is significant, as p110d expression is restricted to hematopoietic cells and p110d bears transforming properties independent of Ras.While previously published work indicates that the PI3K p110a and p110d inhibitor, GDC-0941, inhibits proliferation of GOF Shp2-expressing cells, we tested if the potent p110d-specific inhibitor, GS-9820, is similarly effective. GOF Shp2-expressing bone marrow LDMNCs treated with GS-9820 demonstrated significantly reduced proliferation in a dose-dependent fashion, while GS-9820 failed to inhibit the proliferation of WT Shp2-expressing cells. GS-9820 treatment decreased Akt phosphorylation (S473 and T308) as well as reduced Erk phosphorylation, indicating that p110d inhibition also reduces signaling within the Ras-MEK-Erk pathway. While PI3K activates the canonical Akt-mTORC1 pathway, it also positively feeds back to the Ras-MEK-Erk pathway via activation of Rac-Pak-MEK; therefore, we evaluated if p110d inhibition adds to or is redundant with MEK inhibition. Treatment of GOF Shp2-expressing hematopoietic cells with the MEK inhibitor, PD0325901, effectively reduced proliferation, and addition of GS-9820 further significantly reduced proliferation, indicating that p110d works cooperatively with MEK to promote GOF Shp2-induced disease.Collectively, our findings suggest that PI3K catalytic subunit p110d functions in a Ras-MEK-Erk pathway-independent manner to promote GOF Shp2-induced hypersensitivity to GM-CSF, and suggest that PI3K p110d inhibition in combination with MEK inhibition may be a novel, optimal approach for the treatment of JMML. Disclosures:No relevant conflicts of interest to declare.

Overexpression Of DNMT3a and DNMT3b Is Related To Downregulation Of Mir-29a In Juvenile Myelomonocytic Leukemia (JMML)

Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. This disease is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS, NF1, PTPN11, and CBL have been linked to be responsible for the pathogenesis of JMML in up to 85% of cases. Treatment is very difficult in JMML, and only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28-55%), with 5-year disease-free survival rates of 25-40%. JMML occurs in an age-range when genes are actively being turned on or off in children in adaption to the oxygenized environment after birth. Epigenetics plays a key role in this developmental plasticity. We previously reported hypermethylation on the promoter of PTEN in 77% of JMML patients, and decitabine, a DNA-hypomethylating reagent, significantly inhibited colony formation (CFU-GM) in JMML cells in vitro. In addition, other groups found that aberrant DNA methylation on promoters of BMP4, CALCA, CDKN2B, and RARB is significantly associated with poor prognosis in JMML. Taking together, these data suggest that epigenetic mechanisms may contribute to the pathogenesis of JMML.MicroRNAs (miRNAs) have been reported to play an important role in myeloid differentiation and activation. miRNA function is highly dependent on the cell type. Recently, we reported that miR-183 is overexpressed in JMML. Other groups have reported aberrant expression of miR-29a in acute myeloid leukemia and other cancers. Both miR-183 and miR-29a are located on chromosome 7q32 in humans, which is frequently disrupted in JMML. We hypothesized that miR-29a may be deregulated in JMML, and contribute to the aberrant epigenetic regulation in JMML.In order to test our hypothesis, we collected peripheral blood or bone marrow from 41 JMML patients and 14 normal individuals. Total RNAs were extracted from mononuclear cells (MNCs) using Trizol. We first evaluated the expression levels of miR-29a by using relative-quantitative real-time RT-PCR (qRT-PCR). We found that the expression levels (RQ) of miR-29a in patients are significantly lower than that in normal individuals (median 0.45 vs. 1.11, p< 0.001). By analysis of the expression levels of miR-29a together with previous data on miR-183 in these JMML patients and normal controls, we found that the RQ of miR-29a is inversely correlated with RQ of miR-183 (Spearman's r=-51, p<0.001). This suggests that the expression of miR-29a is segregated from that of miR-183 in MNCs, although they are located only 1.1 million base-pairs apart on chromosome 7q32. When further evaluating the mRNA expression levels of miR-29a targeting genes by using qRT-PCR, we found that RQ of DNA methyltransferases 3A and 3B (DNMT3a and DNMT3b) were significantly increased in JMML patients' samples in comparison with normal controls (median 5.16 vs. 1.00, p=0.001 for DNMT3a; median 3.45 vs. 1.08, p=0.001). Strikingly, the RQ of miR-29a was inversely correlated with the RQs of both DNMTs (Spearman's r=-0.62 for DNMT3a; Spearman's r=-0.79 for DNMT3b, p<0.001), which suggests that miR-29a plays a significant role in the regulation of DNMT 3a and DNMT3b in MNCs. In conclusion, we found that overexpression of DNMT3a and DNMT3b, the two key molecules in DNA methylation of epigenetic modification in developmental plasticity, is related to downregulation of miR-29a in JMML. In addition to our previous report on overexpression of miR-183 in JMML, this finding provides new insights into the role of miRNAs in the aberrant epigenetic regulation in JMML, and it may apply to other pediatric malignancies. Further investigation is ongoing to uncover the mechanism of down-regulated miR-29a in JMML. If the underexpression of miR-29a in JMML is due to the hypermethylation on miR-29a promoter, as reported in prostate cancer cells, demethylating agents, such as decitabine, or soybean products, isoflavone, could be potential drugs to treat JMML. Disclosures:No relevant conflicts of interest to declare.

Deficiency of CREB and over expression of miR-183 in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare leukemia of infancy and early childhood. A hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro, but have normal sensitivity to IL-3, which shares the β-subunit receptor with GM-CSF.1, 2 The pathogenesis of JMML is linked to dysregulated signal transduction through the Ras signaling pathway caused by mutations of RAS, PTPN11 and c-CBL, or loss-of-heterozygosity of NF1.3, 4 Although most patients with JMML experience an aggressive disease course, some others spontaneously resolve.3, 5 No specific mutation has been definitively demonstrated to confer a poorer prognosis, with the exception of perhaps PTPN11, which has been shown to be associated with a higher risk of relapse in some series. In addition, gene expression and methylation analyses have identified signatures associated with outcome that are unexpectedly independent of genotype,5, 6 leading us to believe that additional mutations or events mitigate the course of JMML. Finally, the mechanisms that underlie selective GM-CSF hypersensitivity are not well established in JMML.

Whole Exome Analysis Reveals Spectrum of Gene Mutations in Juvenile Myelomonocytic Leukemia

AbstractAbstract 170 Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods:We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation.Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers’ protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results:For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases.Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion:Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

Mir-183 Over-Expression: A Potential Biomarker for Juvenile Myelomonocytic Leukemia (JMML).

Abstract 2558▪▪This icon denotes a clinically relevant abstractJuvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p<0.001); but the median level of miR-34b was only slightly higher in JMML subjects, and not significantly so, compared to normal individuals (median=1.4 vs 1.0, p>0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p<0.05). Based on a robust regression analysis, for every unit increase in the square root of RQ miR-183, the monocyte percentage significantly increased by 0.73% (SE=0.32%, p=0.023). This is the first evidence suggesting that microRNAs may contribute to the pathogenesis of JMML. miR-183 may also serve as an important biomarker that can be directly and quantitatively linked to significant clinical parameters in JMML. It also may ultimately provide a target for JMML therapy. Disclosures:No relevant conflicts of interest to declare.

Current Management of Juvenile Myelomonocytic Leukemia and the Impact of RAS Mutations

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myelodysplastic/myeloproliferative disorder that affects young children. It is characterized by hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. The pathogenesis of JMML seems to arise from constitutional activation of the GM-CSF/RAS (a GTPase) signaling pathway, a result of mutations in RAS, NF1, PTPN11, and CBL that interfere with downstream components of the pathway. Most patients with JMML usually experience an aggressive clinical course, and hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment, although the high rates of relapses and graft failures are of great concern. In contrast, a certain proportion of patients experience a stable clinical course for a considerable period of time, and sometimes the disease even spontaneously resolves without any treatment. Recent studies have provided us with increased knowledge of genotype-phenotype correlations in JMML, and suggested that differences in clinical courses may reflect genetic status. Thus, genotype-based management is of current international interest, especially for JMML with RAS mutations. Cumulative evidence suggests that RAS mutations can be related to favorable clinical outcomes, and HSCT may not have to be a mandatory therapeutic option for a portion of patients with this mutation, although a consensus regarding genotype-based management has not yet been achieved. Further efforts toward identifying which patients who will do well without HSCT are required.

Novel splicing-factor mutations in juvenile myelomonocytic leukemia

Novel splicing-factor mutations in juvenile myelomonocytic leukemia