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Mantle cell lymphoma (MCL) is a rare subtype of non‐Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR‐S1 (a close analog of the clinical‐stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib‐resistant MCL patient–derived xenograft (PDX) mouse model, OR‐S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2‐specific inhibitor GSK126, OR‐S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR‐S1–sensitive or insensitive MCL cell lines and showed that OR‐S1 treatment modulated B‐cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib‐resistant MCL via CDKN1C‐mediated cell cycle arrest., This study reveals that oral administration of OR‐S1 significantly suppressed tumor growth in an ibrutinib‐resistant mantle cell lymphoma (MCL) patient–derived xenograft mouse model. OR‐S1 induces cell cycle arrest via the upregulation of CDKN1C. CDKN1C expression was directly regulated by enhancer of zeste homologs 1 and 2 (EZH1/2).

Chronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115+ Ly6Chigh monocytes (an equivalent subpopulation to human classical CD14+ CD16− monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1–mediated transcriptional signature in human CD34+ cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased HOXA9 gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.

Recent progress in high-speed sequencing technology has revealed that tumors harbor novel mutations in a variety of genes including those for molecules involved in epigenetics and splicing, some of which were not categorized to previously thought malignancy-related genes. However, despite thorough identification of mutations in solid tumors and hematological malignancies, how these mutations induce cell transformation still remains elusive. In addition, each tumor usually contains multiple mutations or sometimes consists of multiple clones, which makes functional analysis difficult. Fifteen years ago, it was proposed that combination of two types of mutations induce acute leukemia; Class I mutations induce cell growth or inhibit apoptosis while class II mutations block differentiation, co-operating in inducing acute leukemia. This notion has been proven using a variety of mouse models, however most of recently found mutations are not typical class I/II mutations. Although some novel mutations have been found to functionally work as class I or II mutation in leukemogenesis, the classical class I/II theory seems to be too simple to explain the whole story. We here overview the molecular basis of hematological malignancies based on clinical and experimental results, and propose a new working hypothesis for leukemogenesis.

Key Points RUNX1a, but not RUNX1b, is overexpressed in CD34+ cells from patients with myelodysplastic/myeloproliferative neoplasms. SRSF2P95H mutation induces RUNX1a overexpression and a monocytic phenotype in TF-1 cells.

Myeloid malignancies consist of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and myeloproliferative neoplasm (MPN). The latter two diseases have preleukemic features and frequently evolve to AML. As with solid tumors, multiple mutations are required for leukemogenesis. A decade ago, these gene alterations were subdivided into two categories: class I mutations stimulating cell growth or inhibiting apoptosis; and class II mutations that hamper differentiation of hematopoietic cells. In mouse models, class I mutations such as the Bcr-Abl fusion kinase induce MPN by themselves and some class II mutations such as Runx1 mutations induce MDS. Combinations of class I and class II mutations induce AML in a variety of mouse models. Thus, it was postulated that hematopoietic cells whose differentiation is blocked by class II mutations would autonomously proliferate with class I mutations leading to the development of leukemia. Recent progress in high-speed sequencing has enabled efficient identification of novel mutations in a variety of molecules including epigenetic factors, splicing factors, signaling molecules and proteins in the cohesin complex; most of these are not categorized as either class I or class II mutations. The functional consequences of these mutations are now being extensively investigated. In this article, we will review the molecular basis of hematological malignancies, focusing on mouse models and the interfaces between these models and clinical findings, and revisit the classical class I/II hypothesis.

Activating mutations of c-Kit are frequently found in acute myeloid leukemia (AML) patients harboring t(8;21) chromosomal translocation generating a fusion protein AML1-ETO. Here we show that an active mutant of c-Kit cooperates with AML1-ETO to induce AML in mouse bone marrow transplantation models. Leukemic cells expressing AML1-ETO with c-Kit(D814V) were serially transplantable. Transplantation experiments indicated that lineage(-)c-Kit(+)Sca-1(+) (KSL) leukemic cells, but not lineage(+) leukemic cells, were enriched for leukemia stem cells (LSCs). Comparison of gene expression profiles between KSL leukemic and normal cells delineated that CD200R1 was highly expressed in KSL leukemic cells as compared with KSL normal cells. Upregulation of CD200R1 was verified in lineage(-) leukemic cells, but not in lineage(+) leukemic cells. CD200R1 expression in the lineage(-) leukemic cells was not correlated with the frequency of LSCs, indicating that CD200R1 is not a useful marker for LSCs in these models. Interestingly, CD200R1 was upregulated in KSL cells transduced with AML1-ETO, but not with other leukemogenic mutants, including c-Kit(D814V), AML1(D171N), and AML1(S291fsX300). Consistently, upregulation of CD200R1 in lineage(-) leukemic cells was observed only in the BM of mice suffering from AML1-ETO-positive leukemia. In conclusion, AML1-ETO upregulated CD200R1 in lineage(-) cells, which was characteristic of AML1-ETO-positive leukemia in mice.

The Bcr-Abl oncogene causes human Philadelphia chromosome-positive (Ph(+)) leukemias, including B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) with chronic phase (CML-CP) to blast crisis (CML-BC). Previous studies have demonstrated that Src family kinases are required for the induction of B-ALL, but not for CML, which is induced by Bcr-Abl in mice. In contrast, it has been reported that Fyn is up-regulated in human CML-BC compared with CML-CP, implicating Fyn in the blast crisis transition. Here, we aimed to delineate the exact role of Fyn in the induction/progression of Ph(+) leukemias. We found that Fyn is expressed in mouse hematopoietic cells at varying stages of development, including c-kit(+)Sca-1(+)Lin(-) cells. Notably, Fyn is highly expressed in some of human lymphomas, but not in human Ph(+) leukemias including CML-BC. In mouse bone marrow transplantation models, mice transplanted with wild-type or Fyn-deficient bone marrow cells transduced with Bcr-Abl showed no differences in the development of B-ALL or CML-like diseases. Similarly, Fyn deficiency failed to impact the development of myeloid CML-BC induced by Bcr-Abl and Hes1. Elevated expression of Fyn was not found in mouse samples of Bcr-Abl-mediated CML- and CML-BC-like diseases. Thus, Fyn is not required for the pathogenesis of Bcr-Abl-mediated leukemias.

It is well established that Peyer’s patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM+B220+ B cells to IgA+B220+ B cells, but recovered on IgA+B220− plasmablasts for their emigration from the PPs. Thus, IgA+B220− plasmablasts migrated in response to S1P in vitro. Additionally, IgA+ plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA+ plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG–dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)+/− hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc+ cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy. Cancer Res; 75(10); 2005–16. ©2015 AACR.

High levels of HES1 expression are frequently found in BCR-ABL(+) chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC-like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC-like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyer's patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes -stimulated CD11c + dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-β, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c + DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system. The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

Recurrent mutations of ASXL1 (Additional sex combs-like1) are found in various hematological malignancies including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, and acute myeloid leukemia (AML) with myelodysplasia-related changes. Additionally, ASXL1 mutations are linked with adverse survival in a variety of myeloid malignancies. A previous study demonstrated that loss of ASXL1 in mice promotes myeloid transformation by impairing polycomb repressive complex 2 (PRC2)-mediated gene repression at a number of critical loci and leads to myeloid transformation. However, most ASXL1 mutations are heterozygous and located in the 5’ region of the last exon, indicating a dominant-negative or gain-of-function feature of a truncated ASXL1 protein. Therefore, we investigated if the C-terminal truncated form of ASXL1 (ASXL1-MT) contributes to the development of myeloid malignancies. To this end, we examined the effects of ASXL1-MT using in vitro and in vivo experiments. In in vitro experiment, expression of ASXL1-MT inhibited G-CSF-induced myeloid differentiation of 32Dcl3 cells. In a mouse bone marrow transplantation (BMT) model, ASXL1-MT induced multilineage dysplasia, differentiation block, slowly progressive pancytopenia, BM hyperplasia and splenomegaly. The transduced mice died of severe anemia after a long latency (median survival, 400.5 days), and some of the mice progressed to overt leukemia. Thus, the current model displays all of the features of human MDS. In addition, ASXL1-MT collaborated with N-RAS-G12V, which confers a proliferative advantage, in inducing progression of N-RAS-G12V-induced myeloproliferative neoplasm (MPN) to AML, suggesting that ASXL1-MT contributes to leukemic transformation by inhibiting differentiation of MPN cells. To clarify the molecular mechanism for differentiation block and MDS development induced by ASXL1-MT, we performed expression profiles of 32Dcl3 cells transduced with ASXL1-MT and BM cells of the MDS mice. Of note, gene set enrichment analysis (GSEA) of BM cells of the MDS mice indicated that ASXL1-MT induced an expression profile which inversely correlated with known PRC target genes. In fact, ASXL1-MT remarkably derepressed expression of posterior Hoxa genes, including Hoxa5, Hoxa9 and Hoxa10, which are epigenetically silenced by PRC2 in mature cells. In consistent with this, H3K27me3 was globally reduced in ASXL1-MT transduced cells. We also found ASXL1-MT as well as wild type ASXL1 (ASXL1-WT) can bind to EZH2 and, importantly, co-expression of ASXL1-MT with ASXL1-WT efficiently inhibited the binding between ASXL1-WT and EZH2, suggesting a dominant-negative role of ASXL1-MT against the PRC2 function. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that H3K27me3 and Ezh2-bindig profoundly decreased around the promoter regions of Hoxa5, Hoxa9, and Hoxa10 in the MDS mice, correlating with the upregulation of their mRNA expression. On the other hand, we found that ASXL1-MT reduced the expression of Clec5a, a type 2 transmembrane receptor and that this reduction was associated with differentiation block of the 32Dcl3 cells. Moreover, utilizing an shRNA or a mutant form of Clec5a, we identified that Clec5a plays essential roles in myeloid differentiation of 32Dcl3 cells. Lastly, we searched for microRNAs deregulated by ASXL1-MT since a large subset of microRNAs are found to be transcriptionally regulated by PRC2. Among upregulated microRNAs related to myeloid malignancies, we found that miR-125a targeted 3’UTR of Clec5a gene, repressed Clec5a expression and inhibited granulocytic differentiation in vitro. Intriguingly, H3K27me3 and Ezh2-bindig greatly decreased around the miR-125a gene in the BM cells of the MDS mice, similar to the results of ChIP assays around Hoxa genes. The present results indicate that ASXL1-MT which results in a truncated protein product may (1) inhibit PRC2-function by impairing the interaction of EZH2 with the ASXL1-WT and (2) promote myeloid transformation through impaired PRC2-mediated repression of posterior HOXAs and miR-125a, and subsequent suppression of CLEC5A. HOXA9 and CLEC5A expression were shown to be high and low, respectively, in MDS patients with ASXL1-MT. Our data provide evidence for a novel axis in MDS pathogenesis and implicate both mutant forms of ASXL1 and miR-125a as therapeutic targets in MDS. Disclosures: No relevant conflicts of interest to declare.