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Transcription and pre-mRNA splicing are key steps in the control of gene expression and mutations in genes regulating each of these processes are common in leukaemia.sup.1,2. Despite the frequent overlap of mutations affecting epigenetic regulation and splicing in leukaemia, how these processes influence one another to promote leukaemogenesis is not understood and, to our knowledge, there is no functional evidence that mutations in RNA splicing factors initiate leukaemia. Here, through analyses of transcriptomes from 982 patients with acute myeloid leukaemia, we identified frequent overlap of mutations in IDH2 and SRSF2 that together promote leukaemogenesis through coordinated effects on the epigenome and RNA splicing. Whereas mutations in either IDH2 or SRSF2 imparted distinct splicing changes, co-expression of mutant IDH2 altered the splicing effects of mutant SRSF2 and resulted in more profound splicing changes than either mutation alone. Consistent with this, co-expression of mutant IDH2 and SRSF2 resulted in lethal myelodysplasia with proliferative features in vivo and enhanced self-renewal in a manner not observed with either mutation alone. IDH2 and SRSF2 double-mutant cells exhibited aberrant splicing and reduced expression of INTS3, a member of the integrator complex.sup.3, concordant with increased stalling of RNA polymerase II (RNAPII). Aberrant INTS3 splicing contributed to leukaemogenesis in concert with mutant IDH2 and was dependent on mutant SRSF2 binding to cis elements in INTS3 mRNA and increased DNA methylation of INTS3. These data identify a pathogenic crosstalk between altered epigenetic state and splicing in a subset of leukaemias, provide functional evidence that mutations in splicing factors drive myeloid malignancy development, and identify spliceosomal changes as a mediator of IDH2-mutant leukaemogenesis. Analyses of transcriptomes from patients with acute myeloid leukaemia identified frequently co-occurring mutations of IDH2 and SRSF2, which functional analyses showed to have distinct and coordinated leukaemogenic effects on the epigenome and RNA splicing., Author(s): Akihide Yoshimi [sup.1] , Kuan-Ting Lin [sup.2] , Daniel H. Wiseman [sup.3] [sup.4] , Mohammad Alinoor Rahman [sup.2] , Alessandro Pastore [sup.1] , Bo Wang [sup.1] , Stanley Chun-Wei [...]

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition., Introduction Fusions involving neurotrophic receptor tyrosine kinases (NTRKs) were among the first gene translocations described in cancer (1, 2). NTRK1, NTRK2, and NTRK3 (encoding TrkA, TrkB, and TrkC, respectively; hereafter [...]

Author(s): Michael Seiler [1]; Akihide Yoshimi [2]; Rachel Darman [1]; Betty Chan [1]; Gregg Keaney [1]; Michael Thomas [1]; Anant A Agrawal [1]; Benjamin Caleb [1]; Alfredo Csibi [1]; Eckley [...]

Author(s): Stanley Chun-Wei Lee [1]; Heidi Dvinge [2, 3]; Eunhee Kim [1]; Hana Cho [1]; Jean-Baptiste Micol [1]; Young Rock Chung [1]; Benjamin H Durham [1]; Akihide Yoshimi [1]; Young [...]

Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (1-3). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition (4-6). Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 ([Srsf2.sup.P95H])--which commonly occurs in individuals with MDS and AML--in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AML., Mutations in the spliceosomal genes SRSF2, U2AF1, and SF3B1 are the most common class of mutations in patients with MDS (1-3) and occur across the entire spectrum of myeloid malignancies, [...]

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBo and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs., Introduction Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy characterized by a relentless proliferation of immature myeloid blasts. Recent studies have demonstrated that the apparently uniform leukemia cell [...]

Author(s): Stanley Chun-Wei Lee; Heidi Dvinge; Eunhee Kim; Hana Cho; Jean-Baptiste Micol; Young Rock Chung; Benjamin H Durham; Akihide Yoshimi; Young Joon Kim; Michael Thomas; Camille Lobry; Chun-Wei Chen; Alessandro [...]