DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers.

Cancer immune evasion contributes to checkpoint immunotherapy failure in many patients with metastatic cancers. The embryonic transcription factor DUX4 was recently characterized as a suppressor of interferon-γ signaling and antigen presentation that is aberrantly expressed in a small subset of primary tumors. Here, we report that DUX4 expression is a common feature of metastatic tumors, with ~10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressing DUX4. DUX4 expression is significantly associated with immune cell exclusion and decreased objective response to PD-L1 blockade in a large cohort of urothelial carcinoma patients. DUX4 expression is a significant predictor of survival even after accounting for tumor mutational burden and other molecular and clinical features in this cohort, with DUX4 expression associated with a median reduction in survival of over 1 year. Our data motivate future attempts to develop DUX4 as a biomarker and therapeutic target for checkpoint immunotherapy resistance.

Over time cancer patients can become resistant to traditional treatments such as chemotherapy and radiotherapy. In some cases, this can be counteracted by administering a new type of treatment called immune checkpoint inhibition which harnesses a patient's own immune system to eradicate the tumor. However, a significant proportion of cancers remain resistant, even when these immunotherapy drugs are used. This is potentially caused by tumors reactivating a gene called DUX4, which is briefly turned on in the early embryo shortly after fertilization, but suppressed in healthy adults. Activation of DUX4 during the early stages of cancer has been shown to remove the cell surface proteins the immune system uses to recognize tumors. However, it remained unclear whether DUX4 changes the response to immunotherapy in more advanced cancers which have begun to spread and metastasize to other parts of the body. To investigate, Pineda and Bradley analyzed publicly available sequencing data which revealed the genes turned on and off in patients with different types of cancer. The analysis showed that DUX4 is reactivated in approximately 10­50% of advanced bladder, breast, kidney, prostate and skin cancers. Next, Pineda and Bradley studied a cohort of patients with advanced bladder cancer who had been treated with immune checkpoint inhibitors. They found that patients with tumors in which DUX4 had been turned back on had shorter survival times than patients who had not reactivated the gene. These results suggest that the activity of DUX4 could be used to predict which patients with advanced bladder cancer may benefit from immune checkpoint inhibitors. In the future, this work could be extended to see if DUX4 could be used as a prognostic tool for other types of cancer. Future studies could also investigate if the DUX4 gene could be a therapeutic target for mitigating resistance to immunotherapy in metastatic cancers.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

DUX4 is a common driver of immune evasion and immunotherapy failure in metastatic cancers.

Cancer immune evasion contributes to checkpoint immunotherapy failure in many patients with metastatic cancers. The embryonic transcription factor DUX4 was recently characterized as a suppressor of interferon-γ signaling and antigen presentation that is aberrantly expressed in a small subset of primary tumors. Here, we report that DUX4 expression is a common feature of metastatic tumors, with ~10-50% of advanced bladder, breast, kidney, prostate, and skin cancers expressing DUX4. DUX4 expression is significantly associated with immune cell exclusion and decreased objective response to PD-L1 blockade in a large cohort of urothelial carcinoma patients. DUX4 expression is a significant predictor of survival even after accounting for tumor mutational burden and other molecular and clinical features in this cohort, with DUX4 expression associated with a median reduction in survival of over one year. Our data motivate future attempts to develop DUX4 as a biomarker and therapeutic target for checkpoint immunotherapy resistance.

The proto-oncogene SRC phosphorylates cGAS to inhibit an antitumor immune response.

Cyclic GMP-AMP synthase (cGAS) is a DNA sensor and responsible for inducing an antitumor immune response. Recent studies reveal that cGAS is frequently inhibited in cancer, and therapeutic targets to promote antitumor cGAS function remain elusive. SRC is a proto-oncogene tyrosine kinase and is expressed at elevated levels in numerous cancers. Here, we demonstrate that SRC expression in primary and metastatic bladder cancer negatively correlates with innate immune gene expression and immune cell infiltration. We determine that SRC restricts cGAS signaling in human cell lines through SRC small molecule inhibitors, depletion, and overexpression. cGAS and SRC interact in cells and in vitro, while SRC directly inhibits cGAS enzymatic activity and DNA binding in a kinase-dependent manner. SRC phosphorylates cGAS, and inhibition of cGAS Y248 phosphorylation partially reduces SRC inhibition. Collectively, our study demonstrates that cGAS antitumor signaling is hindered by the proto-oncogene SRC and describes how cancer-associated proteins can regulate the innate immune system.

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.

Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells.

Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus-thymidine kinase (HSV-TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV-TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy.

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.

Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia.

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

Most human introns are recognized via multiple and tissue-specific branchpoints.

Although branchpoint recognition is an essential component of intron excision during the RNA splicing process, the branchpoint itself is frequently assumed to be a basal, rather than regulatory, sequence feature. However, this assumption has not been systematically tested due to the technical difficulty of identifying branchpoints and quantifying their usage. Here, we analyzed ∼1.31 trillion reads from 17,164 RNA sequencing data sets to demonstrate that almost all human introns contain multiple branchpoints. This complexity holds even for constitutive introns, 95% of which contain multiple branchpoints, with an estimated five to six branchpoints per intron. Introns upstream of the highly regulated ultraconserved poison exons of SR genes contain twice as many branchpoints as the genomic average. Approximately three-quarters of constitutive introns exhibit tissue-specific branchpoint usage. In an extreme example, we observed a complete switch in branchpoint usage in the well-studied first intron of HBB (ß-globin) in normal bone marrow versus metastatic prostate cancer samples. Our results indicate that the recognition of most introns is unexpectedly complex and tissue-specific and suggest that alternative splicing catalysis typifies the majority of introns even in the absence of differences in the mature mRNA.