Systematic evaluation of AML-associated antigens identifies anti-U5 SNRNP200 therapeutic antibodies for the treatment of acute myeloid leukemia.

Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.

Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments.

Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation.

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation. SIGNIFICANCE: Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221.

Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias.

Single-cell genomics reveals the genetic and molecular bases for escape from mutational epistasis in myeloid neoplasms.

Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are among the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple-concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. In this study, we performed bulk and single-cell analyses of patients with myeloid malignancy who were harboring ≥2 splicing factor mutations, to understand the frequency and basis for the coexistence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4231 patients (q < .001), 0.85% harbored 2 concomitant bona fide splicing factor mutations, ∼50% of which were present in the same individual cells. However, the distribution of mutations in patients with double mutations deviated from that in those with single mutations, with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in those with double-mutations had reduced effects on RNA splicing and/or binding compared with the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating the molecular effects of splicing factor mutations as well as their cooccurrences/exclusivities with one another.

Aberrant RNA Splicing in Cancer.

RNA splicing, the enzymatic process of removing segments of premature RNA to produce mature RNA, is a key mediator of proteome diversity and regulator of gene expression. Increased systematic sequencing of the genome and transcriptome of cancers has identified a variety of means by which RNA splicing is altered in cancer relative to normal cells. These findings, in combination with the discovery of recurrent change-of-function mutations in splicing factors in a variety of cancers, suggest that alterations in splicing are drivers of tumorigenesis. Greater characterization of altered splicing in cancer parallels increasing efforts to pharmacologically perturb splicing and early-phase clinical development of small molecules that disrupt splicing in patients with cancer. Here we review recent studies of global changes in splicing in cancer, splicing regulation of mitogenic pathways critical in cancer transformation, and efforts to therapeutically target splicing in cancer.

A Case of Lyme Carditis Presenting with Atrial Fibrillation.

We report a case of a 20-year-old man who presented to our institution with a new arrhythmia on a routine EKG. Serial EKG tracings revealed various abnormal rhythms such as episodes of atrial fibrillation, profound first degree AV block, and type I second degree AV block. He was found to have positive serologies for Borrelia burgdorferi. After initiation of antibiotic therapy, the atrial arrhythmias and AV block resolved. Here, we present a case of Lyme carditis presenting with atrial fibrillation, a highly unusual presentation of Lyme carditis.

Measurement of BH3-only protein tolerance.

The BCL2 family of proteins regulates cellular life and death decisions. Among BCL2 family members, BH3-only proteins have critical roles by neutralizing antiapoptotic family members, as well as directly activating BAX and BAK. Despite widespread occurrence of BH3-only protein upregulation in response to various stresses, this process is rarely quantified. Moreover, it is unclear whether all BH3-only proteins are equipotent at inducing cell death. Here we show that BH3-only proteins increase as much as 15- to 20-fold after various treatments and define a parameter, termed BH3-only tolerance, which measures how many copies of a particular BH3-only protein can be expressed before the majority of cells in a population undergo apoptosis. We not only assess the relative contributions of anti- and proapoptotic BCL2 family members to BH3-only tolerance, but also illustrate how the study of this parameter can be used to understand cellular sensitivity to anticancer drugs and new combinations. These observations provide a new quantitative framework for assessing apoptotic susceptibility under various conditions.

Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo.

Current understanding suggests that malignant stem and progenitor cells must be reduced or eliminated for prolonged remissions in myeloid neoplasms such as acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). Multicolor flow cytometry has been widely used to distinguish stem and myeloid progenitor cells from other populations in normal and malignant bone marrow. In this study, we present a method for assessing drug sensitivity in MDS and AML patient hematopoietic stem and myeloid progenitor cell populations ex vivo using the investigational Nedd8-activating enzyme inhibitor MLN4924 and standard-of-care agent cytarabine as examples. Utilizing a multicolor flow cytometry antibody panel for identification of hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors present in mononuclear cell fractions isolated from bone marrow aspirates, we compare stem and progenitor cell counts after treatment for 24 hours with drug versus diluent. We demonstrate that MLN4924 exerts a cytotoxic effect on MDS and AML stem and progenitor cell populations, whereas cytarabine has more limited effects. Further application of this method for evaluating drug effects on these populations ex vivo and in vivo may inform rational design and selection of therapies in the clinical setting. Stem Cells Translational Medicine 2017;6:840-850.

4EBP1/c-MYC/PUMA and NF-κB/EGR1/BIM pathways underlie cytotoxicity of mTOR dual inhibitors in malignant lymphoid cells.

The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in multiple malignancies. Rapamycin analogs, which partially inhibit mTOR complex 1 (mTORC1), exhibit immunosuppressive and limited antitumor activity, but sometimes activate survival pathways through feedback mechanisms involving mTORC2. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR active site. Here we show that disruption of either mTOR-containing complex is toxic to acute lymphocytic leukemia (ALL) cells and identify 2 previously unrecognized pathways leading to this cell death. Inhibition of mTORC1-mediated 4EBP1 phosphorylation leads to decreased expression of c-MYC and subsequent upregulation of the proapoptotic BCL2 family member PUMA, whereas inhibition of mTORC2 results in nuclear factor-κB-mediated expression of the Early Growth Response 1 (EGR1) gene, which encodes a transcription factor that binds and transactivates the proapoptotic BCL2L11 locus encoding BIM. Importantly, 1 or both pathways contribute to death of malignant lymphoid cells after treatment with dual mTORC1/mTORC2 inhibitors. Collectively, these observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors in ALL.

Emerging understanding of Bcl-2 biology: Implications for neoplastic progression and treatment.

Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that anti-apoptotic Bcl-2 family members modulate the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions.