Stanley Chun-Wei Lee

BET inhibitor resistance emerges from leukaemia stem cells

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL–AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.

Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins.

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). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition. 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 (Srsf2P95H)—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.

Diverse and Targetable Kinase Alterations Drive Histiocytic Neoplasms

UNLABELLED Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAF(V600E) mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAF(V600E)-wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAF(V600E)-wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. SIGNIFICANCE We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders.

Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression

One of the most common analysis tasks in genomic research is to identify genes that are differentially expressed (DE) between experimental conditions. Empirical Bayes (EB) statistical tests using moderated genewise variances have been very effective for this purpose, especially when the number of biological replicate samples is small. The EB procedures can however be heavily influenced by a small number of genes with very large or very small variances. This article improves the differential expression tests by robustifying the hyperparameter estimation procedure. The robust procedure has the effect of decreasing the informativeness of the prior distribution for outlier genes while increasing its informativeness for other genes. This effect has the double benefit of reducing the chance that hypervariable genes will be spuriously identified as DE while increasing statistical power for the main body of genes. The robust EB algorithm is fast and numerically stable. The procedure allows exact small-sample null distributions for the test statistics and reduces exactly to the original EB procedure when no outlier genes are present. Simulations show that the robustified tests have similar performance to the original tests in the absence of outlier genes but have greater power and robustness when outliers are present. The article includes case studies for which the robust method correctly identifies and downweights genes associated with hidden covariates and detects more genes likely to be scientifically relevant to the experimental conditions. The new procedure is implemented in the limma software package freely available from the Bioconductor repository.

Therapeutic targeting of splicing in cancer

Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein–RNA interactions in cancer.

Polycomb repressive complex 2 component Suz12 is required for hematopoietic stem cell function and lymphopoiesis.

Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context.

Recurrent CDKN1B (p27) mutations in hairy cell leukemia.

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 CDKN1B in 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 CDKN1B mutation was clonal, implying an early role of CDKN1B mutations in the pathogenesis of HCL. CDKN1B mutations were not found to impact clinical characteristics or outcome in this cohort. These data identify HCL as having the highest frequency of CDKN1B mutations among cancers and identify CDNK1B as 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 CDKN1B mutations.

Epigenetic Regulator Smchd1 Functions As a Tumor Suppressor

SMCHD1 is an epigenetic modifier of gene expression that is critical to maintain X chromosome inactivation. Here, we show in mouse that genetic inactivation of Smchd1 accelerates tumorigenesis in male mice. Loss of Smchd1 in transformed mouse embryonic fibroblasts increased tumor growth upon transplantation into immunodeficient nude mice. In addition, loss of Smchd1 in Eμ-Myc transgenic mice that undergo lymphomagenesis reduced disease latency by 50% relative to control animals. In premalignant Eμ-Myc transgenic mice deficient in Smchd1, there was an increase in the number of pre-B cells in the periphery, likely accounting for the accelerated disease in these animals. Global gene expression profiling suggested that Smchd1 normally represses genes activated by MLL chimeric fusion proteins in leukemia, implying that Smchd1 loss may work through the same pathways as overexpressed MLL fusion proteins do in leukemia and lymphoma. Notably, we found that SMCHD1 is underexpressed in many types of human hematopoietic malignancy. Together, our observations collectively highlight a hitherto uncharacterized role for SMCHD1 as a candidate tumor suppressor gene in hematopoietic cancers.

Polycomb repressive complex 2 (PRC2) suppresses Eμ-myc lymphoma.

Deregulation of polycomb group complexes polycomb repressive complex 1 (PRC1) and 2 (PRC2) is associated with human cancers. Although inactivating mutations in PRC2-encoding genes EZH2, EED, and SUZ12 are present in T-cell acute lymphoblastic leukemia and in myeloid malignancies, gain-of-function mutations in EZH2 are frequently observed in B-cell lymphoma, implying disease-dependent effects of individual mutations. We show that, in contrast to PRC1, PRC2 is a tumor suppressor in Eµ-myc lymphomagenesis, because disease onset was accelerated by heterozygosity for Suz12 or by short hairpin RNA-mediated knockdown of Suz12 or Ezh2. Accelerated lymphomagenesis was associated with increased accumulation of B-lymphoid cells in the absence of effects on apoptosis or cell cycling. However, Suz12-deficient B-lymphoid progenitors exhibit enhanced serial clonogenicity. Thus, PRC2 normally restricts the self-renewal of B-lymphoid progenitors, the disruption of which contributes to lymphomagenesis. This finding provides new insight regarding the functional contribution of mutations in PRC2 in a range of leukemias.

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor–encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.