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Detailed genomic and epigenomic analyses of MECOM(the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drive inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer which upregulates transcription of EVI1. Here we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1which is frequently present in inv(3)/t(3;3) AML and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly co-occurring genomic alteration in inv(3)/t(3;3) patients. SF3B1mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of six amino acids at the 3’ end of the second Zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo.The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1exon 13 to promote usage of a cryptic branch point and aberrant 3’ splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent co-occurrence of SF3B1mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Loss of function mutations in TET2are common in clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). Increased inflammation and stem cell capacity have been reported in Tet2 KOmouse models, but molecular mechanisms and cell type-specific contributions to explain this phenotype are lacking. To create models of CH-like TET2 deficiency, we transplanted Mx1-CRE Tet2 fl/fl(Tet2 KO)or Tet2 fl/fl(both CD45.1 +)in CD45.2 +recipientsfollowing radiation-free conditioning with ACK2 (anti-cKit) antibody injection. Six months post-transplant, we flow sorted CD45.1 +Tet2 KOand Tet2 fl/flLin -Sca-1 +cKit +(LSK) cells, granulocyte-monocyte progenitors (GMP), and monocytes for RNA and assay for transposase-accessible chromatin (ATAC) sequencing. Differential gene expression analysis identified 902 (468 up, 434 down) differentially expressed genes (DEGs) in LSK, 134 (95 up, 39 down) in GMP, and 474 (125 up, 349 down) in monocytes (p adj<0.05). Gene set enrichment analysis (GSEA) identified that the top Hallmark gene sets in Tet2 KOLSK cells were related to MYC targets, oxidative phosphorylation, and E2F targets. In contrast, GMP and monocytes were most enriched for IFN-alpha and gamma gene sets along with IL6/JAK/STAT3 signaling. ATACseq revealed increased accessibility of myeloid-related transcription factor (TF) binding motifs in Tet2 KOLSK cells (CEBPA, PU.1-IRF(ETS:IRF), and AP-1 TF FOSL2(bZIP)), while prediction of cis-regulating regions via GREAT identified downregulated peaks associated with stem cell maintenance and upregulated peaks associated with myeloid mediated immunity. Peaks upregulated in Tet2 KOGMP cells were enriched for the motifs recognized by CEBPE, ISRE(IRF)) and the AP-1 TF JUND, while upregulated peaks in monocytes were enriched for RUNX1 and CEBPA motifs, as well as AP-1 TF FOS::JUNB. GREAT analysis in GMPs identified cis-regulatory regions related to myeloid mediated immunity, while monocytes possessed those associated with biotic stimuli.

Hairy cell leukemia (HCL) is marked by near 100% mutational frequency of BRAFV600E mutations. Recurrent cooperating genetic events that may contribute to HCL pathogenesis or affect the clinical course of HCL are currently not described. Therefore, we performed whole exome sequencing to explore the mutational landscape of purine analog refractory HCL. In addition to the disease-defining BRAFV600E mutations, we identified mutations in EZH2, ARID1A, and recurrent inactivating mutations of the cell cycle inhibitor CDKN1B(p27). Targeted deep sequencing of CDKN1Bin a larger cohort of HCL patients identify deleterious CDKN1B mutations in 16% of patients with HCL (n = 13 of 81). In 11 of 13 patients the CDKN1Bmutation was clonal, implying an early role of CDKN1Bmutations in the pathogenesis of HCL. CDKN1Bmutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1Bmutations among cancers and identify CDNK1Bas the second most common mutated gene in HCL. Moreover, given the known function of CDNK1B, these data suggest a novel role for alterations in regulation of cell cycle and senescence in HCL with CDKN1Bmutations.

The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZgene to CBP, p300, NCOA3, or TIF2in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eμ-Mycmodel of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Mozincreased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Mozhaploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1translocations in leukemia. We demonstrate that MOZ localizes to the Meis1locus in pre–B-cells and maintains Meis1expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.

Fanconi’s anemia is an inherited disorder characterized by multiple congenital abnormalities, chromosome anomalies and irreversible aplastic anemia. Patients, and possibly family members, have been noted to have an increased incidence of leukemia and other tumors. A family with two affected male children, one of whom developed acute myelomonocytic leukemia, is described. Chromosome preparations in both parents showed abnormalities, a previously unreported finding. Fibroblast cultures from both parents and one brother inoculated with Simian Virus 40, a known oncogenic agent, developed increased numbers of transformed colonies, compared with normal control cultures similarly inoculated. The presence of chromosome abnormalities and cellular susceptibility to oncogenic agents appear to be related to an increased risk of malignancy.

Leukocyte alkaline phosphatase activity has been noted to be different in men and women. The mean leukocyte alkaline phosphatase activity for 74 normal men, aged 19 to 60 years, was 23 mg. of phosphorus per 1010 polvmorphonuclear leukocytes per hour. The corresponding mean value for 75 normal young women, age 19-48 years, was 35 (p < .001). No significant differences between boys and girls occurred until the time of puberty. After the menopause, the values for women approached the values for men. Women treated with androgens had lower leukocyte alkaline phosphatase activity than did control women. These results suggest that androgenic hormones inhibit this enzyme, and that other, as yet undefined endocrine influences, also affect its level of activity. In vitro tests with various concentrations of androgens and estrogens failed to provide conclusive evidence of direct effect on leukocytes although some degree of direct inhibition by androgens was suggested. Studies using saponin to effect enzyme release from leukocyte granules did not demonstrate whether the differences between men and women are differences of enzyme release or of content of leukocyte alkaline phosphatase.

Leukocyte alkaline phosphatase was measured by a biochemical method in a group of 58 mongolian idiots. The mean activity found in the 41 children less than 10 years of age was equivalent to 89 mg. of phosphorus per 1010 neutrophilic leukocytes per hour. This was compared to the value of 66 mg. of phosphorus per 1010 neutrophilic leukocytes per hour obtained in a group of 41 control children in the same age group (after correction of the control mean to the same mean age). The difference is significant at the 0.5 per cent level of confidence. This difference was interpreted as confirming (but not proving) the hypothesis that leukocyte alkaline phosphatase formation is controlled by a gene on chromosome number 21—the chromosome for which mongolian idiots are trisomic. The hypothesis arose because of the known deficiency of this enzyme in the leukocytes of patients with chronic granulocytic leukemia, and the known partial deletion of chromosome 21 in this disease. This finding should provide a stimulus to further investigations into the content of alkaline phosphatase in leukocytes, possible polymorphism of leukocyte alkaline phosphatases, linkages with other inherited traits, and relationships between leukocyte and other tissue phosphatases.

1. Combinations of effective agents produce at least an additive increase in complete remission rates over that which can be achieved when the agents are used individually. 2. Patients who do not achieve complete remission with initial treatment have a significantly shorter survival. 3. Alternating MTX and 6-MP at 28-day intervals during remission does not prolong the duration of remission over that of combined concurrent 6-MP and MTX. 4. The administration of folic acid antagonists intrathecally at 28-day intervals during antimetabolite maintained remission did not prolong the duration of remission. Meningeal leukemia, however, occurred significantly less frequently in these patients. 5. The duration of combined 6-MP and MTX maintained remission is not greater than that of 6-MP maintained remission. 6. The toxicity of 6-MP and MTX in combination in patients in remission is additive. The above conclusions were drawn from this comparative study of combinations of chemotherapeutic agents in children with acute lymphocytic leukemia.

Dying leukocytes suspended in normal serum tend to agglutinate spontaneously into a firm "clot." This phenomenon is associated with cell lysis and the formation of long fibrous strands of DNA-protein. It seems likely that nuclear DNA-protein actually combines in some way with a fraction of the serum proteins (possibly mucoprotein). Such leukocyte agglutination is inhibited by serum containing the L.E. cell factor. LAI is associated with nuclear changes which are characteristic of the L.E. cell phenomenon, and it is most probable that it is an expression of the L.E. cell phenomenon. An understanding of the chemistry of LAI may lead to useful information as to the fundamental mechanism of the L.E. cell phenomenon. Without such understanding, LAI has been used to obtain collateral information concerning the L.E. cell phenomenon: namely, to rule out any important role for DNA-ase, and to confirm the importance of blood platelets. It has also been used to prepare "pure cultures" of L.E. bodies for cytochemical study. On a clinical level, LAI may prove a useful adjunct to the standard L.E. cell tests. However, its lower level of sensitivity and occasional "false positive" reactions make it inadequate as a primary test in systemic lupus.

Cells from bone marrow aspirates and liver, kidney and spleen imprints from patients with leukemia and other nonleukemic hematological and non-hematological disorders were tested for reactivity with murine leukemia (Rauscher) virus antibody using the technique of immunofluorescence. The cells of all eleven patients with acute lymphoblastic leukemia and four out of five patients with chronic lymphatic leukemia reacted strongly. In only six of the 15 patients with myelogenous leukemia and approximately one-third of the 47 patients with miscellaneous disorders (Table 1) was strong immunofluorescence demonstrated.

The effect of 6-MP therapy on the duration of remissions induced by adrenal corticosteroids has been studied as a model for testing of new agents. Ninetytwo patients under age 20 entered the study and were accepted for analysis. Sixty-two (67 per cent) had complete or partial remissions induced by corticosteroids. Patients in remission were randomly assigned to maintenance therapy with either 6-MP or placebo. The median duration of 6-MP-maintained complete remissions was 33 weeks and for placebo, 9 weeks. A sequential experimental design was used to analyze remission times while the study was in progress. This resulted in the study being stopped after analysis of the remission times of 21 pairs of patients (42 patients). Overall survival was not significantly different for the two treatment programs, since patients maintained on placebo were treated with 6-MP when relapse occurred. The activity of the known active antileukemic compound 6-MP was readily detected by this experimental design without compromise of optimal survival. Such a design should prove useful for the evaluation of new agents and also permit study of the remission maintenance activity of a compound separately from its remission inducing activity.

A factor derived from blood platelets has been shown to be necessary for the activation of the L. E. cell factor. This L. E. cell cofactor may be identical with the platelet thromboplastic factor. The relation of these findings to occurrence of the L. E. cell phenomenon in vivo and to the occurrence of a coagulation defect in systemic L. E. has been discussed.

Leukocyte alkaline phosphatase was measured by a biochemical method in a group of 58 mongolian idiots. The mean activity found in the 41 children less than 10 years of age was equivalent to 89 mg. of phosphorus per 1010neutrophilic leukocytes per hour. This was compared to the value of 66 mg. of phosphorus per 1010neutrophilic leukocytes per hour obtained in a group of 41 control children in the same age group (after correction of the control mean to the same mean age). The difference is significant at the 0.5 per cent level of confidence.

Genomic analyses across cancer have identified recurrent somatic mutations in XPO1, which encodes a major nuclear cytoplasmic transport protein responsible for exporting a broad range of cargo proteins from the nucleus to the cytoplasm. Specifically, heterozygous mutations at the XPO1 hotspot residue E571 (most frequently E571K) are recurrent in B-cell malignancies including chronic lymphocytic leukemia, primary mediastinal B-cell lymphoma, and Hodgkin lymphoma. Despite the fact that XPO1 is the target of numerous small molecules in clinical development, functional evidence for precisely how altered XPO1 expression or mutations might promote transformation is not well delineated.

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1. Although only 1 patient with an SRSF2mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2. Mutational analysis of the RNA-seq data identified the SRSF2proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2mutant patients also had a co-existing IDH2mutation (and conversely, 50% of IDH2mutant patients had a co-existing SRSF2mutation). These data are similar to recent observation that SRSF2mutations were the most common accompanying mutation in IDH2mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.

Spliceosomal mutations account for the most frequent class of mutations in patients with MDS and CMML, yet the mechanism by which these mutations perform their driver function is not well understood. Moreover, no genetically engineered murine models for expression of spliceosomal gene mutations from their endogenous loci have been reported. Given the genetic heterogeneity of primary patient samples, we generated a model for conditional expression of the commonly occurring SRSF2P95H mutation from the endogenous murine locus of Srsf2. We compared expression of the Srsf2P95H mutation with genetic inactivation of 0, 1 or 2 copies of Srsf2to understand the biological and mechanistic consequences of spliceosomal mutations relative to loss-of-function.

A 19-yr-old female presented with diffuse histiocytic lymphoma after a 15-yr remission of acute lymphoblastic leukemia. All treatment had been discontinued 7 yr before the onset of the lymphoma. The possible relationships of these two neoplasms are discussed.