Background Medulloblastoma (MB) is the most common pediatric brain tumor. Although standard of care treatment generally results in good prognosis, many patients exhibit treatment-associated lifelong disabilities. This outcome could be improved by employing therapies targeting the molecular drivers of this cancer. Attempts to do so in the SONIC HEDGEHOG MB subgroup (SHH-MB) have largely focused on the SHH pathway’s principal activator, SMOOTHENED (SMO). While inhibitors targeting SMO have shown clinical efficacy, recurrence and resistance are frequently noted, likely resulting from mutations in or downstream of SMO. Therefore, identification of novel SHH regulators that act on the pathway’s terminal effectors could be used to overcome or prevent such recurrence. We hypothesized that PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) is one such regulator and investigated its role and potential targeting in SHH-MB. Methods PRMT5 expression in SHH-MB was first evaluated. Knockdown and pharmacological inhibitors of PRMT5 were used in SHH-MB sphere cultures to determine its effect on viability and SHH signaling. GLI1 arginine methylation was then characterized in primary SHH-MB tissue using LC-MS/MS. Finally, PRMT5 inhibitor efficacy was evaluated in vivo. Results PRMT5 is overexpressed in SHH-MB tissue. Furthermore, SHH-MB viability and SHH activity is dependent on PRMT5. We found that GLI1 isolated from SHH-MB tissues is highly methylated, including three PRMT5 sites that affect SHH-MB cell viability. Importantly, tumor growth is decreased and survival increased in mice given PRMT5 inhibitor. Conclusions PRMT5 is a requisite driver of SHH-MB that regulates tumor progression. A clinically relevant PRMT5 inhibitor represents a promising candidate drug for SHH-MB therapy.
The splicing factor gene, U2AF1, is commonly mutated in myeloid neoplasms and occasionally in solid tumors such as lung adenocarcinomas (LUADs), yet the molecular and physiological consequences of U2AF1 mutations are not well understood. Through genome editing of the endogenous locus, we created a common U2AF1 mutant allele (U2AF1-S34F) in an immortalized human bronchial epithelial cell line, and we inactivated the U2AF1-S34F allele in two LUAD cell lines that naturally harbor this mutation. Cells expressing U2AF1-S34F deployed altered splicing of many transcripts, and they displayed a S34F-characteristic sequence pattern at the proximal 3’ splice site, as previously reported. Remarkably, S34F-associated alternative splicing patterns remained unaffected by changes in the amount of mutant U2AF1 protein, as long as the ratio of mutant and wild-type U2AF1 remained constant, as evidenced by RNA interference-mediated knockdown of the expression of both mutant and wild-type alleles. On the other hand, perturbing the ratio of mutant and wild-type U2AF1, either through overexpression or allelic-specific inactivation of wild-type U2AF1, greatly affected S34F-associated splicing. These results suggest that the ratio of mutant and wild-type U2AF1, rather than the absolute amount of either protein, is critical in controlling splicing outcome. Expression of U2AF1-S34F does not cause cell transformation and LUAD cells with endogenous U2AF1-S34F expression are not dependent on the U2AF1-S34F allele. Instead, wild-type U2AF1 is absolutely required for cell survival in the presence of the mutant allele. To study the effect of U2AF1-S34F in vivo, we created transgenic mice carrying a conditional mutant allele, MG-S34F, at the endogenous U2af1 locus. By crossing the MG-S34F mice with mice carrying a Mx1-Cre transgene and treating the progeny with poly IC, we activated MG-S34F by Cre-mediated recombination to express U2af1-S34F in bone marrow cells. This resulted in cytopenia in multiple blood cell lineages. Similar results were observed in recipient mice following non-competitive transplant. Bone marrow cells expressing U2af1-S34F were competitively disadvantaged in repopulating the hematopoietic system in irradiated mice, suggesting defective hematopoietic stem cell function, and we are currently assessing the long-term impact of U2af1-S34F expression on the incidence of myeloid neoplasms. Citation Format: Dennis Fei, Hayley Motowski, Sameer Prasad, Jovian Yu, Robert Bradley, Harold Varmus. Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4163.
Introduction-Dysregulation of the hedgehog signalling pathway has been linked to the development and progression of a variety of different human tumors including cancers of the skin, brain, colon, prostate, blood, and pancreas. We assessed the clinicopathological factors that are potentially related to expression of Gli1, the transcription factor that is thought to be the most reliable marker of hedgehog pathway activation in bladder cancer. Methods-Bladder cancer cases were identified from the New Hampshire State Cancer Registry as histologically confirmed primary bladder cancer diagnosed between January 1, 2002, and July 31, 2004. Immunohistochemical analysis was performed on a tissue microarray to detect Gli1 and p53 expression in these bladder tumors. We computed odds ratios (ORs) and their 95% CIs for Gli1 positivity for pathological category using T category (from TNM), invasiveness, and grade with both the World Health Organization 1973 and World Health Organization International Society of Urological Pathology criteria. We calculated hazard ratios and their 95% CI for Gli1 positivity and recurrence for both Ta-category and invasive bladder tumors (T1+).
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