SC1/PRDM4 recruits PRMT5 to control the timing of neural precursor differentiation in developing neural stem cells

During cortical development, neural stem cells (NSCs) switch from proliferative to neuron-generating asymmetric divisions. Here we investigated the role of Schwann cell factor 1 (SC1/PRDM4), a transcriptional repressor highly expressed in the developing nervous system, during NSCs development. We found that SC1 protein levels were down-regulated in newly differentiating neurons, while remaining high in undifferentiated NSCs, suggesting an asymmetric inheritance of SC1. Knockdown of SC1 in the NSCs led to precocious differentiation of neurons and its overexpression led to an increase in Nestin-expressing precursors. We found that SC1, through its amino-terminus, recruits the chromatin modifier PRMT5, a histone arginine methyltransferase that catalyses histone H4R3 symmetric dimethylation (H4R3me2s). Mutations disrupting SC1/PRMT5 interaction resulted in loss of SC1-mediated increase in undifferentiated neural precursor cells. Our data demonstrate that SC1 and PRMT5 are components of an epigenetic regulatory complex that provides an epigenetic signature of a “stem-like” cellular state in the NSCs and which may be asymmetrically inherited during neurogenic divisions

Abstract 5353: PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC

Abstract Osimertinib is a third-generation EGFR-TKI approved for the first-line treatment of EGFR-driven lung adenocarcinomas. High response rates to osimertinib are observed in patients with activating EGFR mutations. However, there is variability in duration of response and patients eventually develop acquired resistance.Preclinical validation of potential acquired resistance mutations identified in relapsed patients treated with osimertinib is a key challenge to develop new strategies to improve outcomes for patients with EGFR mutation positive NSCLC. Tissue and ctDNA NGS analysis from relapsed patients treated first-line with osimertinib in the FLAURA and ORCHARD trials identified PIK3CA gain of function (GOF 11%) and PTEN loss of function (LOF 5%) mutations.To test whether PIK3CA activating mutations can drive resistance to osimertinib we introduced PIK3CA activating variants (PIK3CA-H1047R and PIK3CA-E453K) or knocked-out PTEN in NSCLC lung cancer cell lines harbouring EGFR activating mutations and WT PIK3CA/PTEN using CRISPR/Cas9 technology. Our results show that PIK3CA GOF and PTEN LOF mutations conferred resistance to osimertinib in vitro. Resistance was associated with increased EC50 for osimertinib and diminished apoptotic response in NSCLC PIK3CA GOF and PTEN LOF mutant CRISPR cell lines when compared to parental cells. Protein analysis by western blot also showed an increase in the basal and osimertinib treated levels of pAKT and pS6 in PIK3CA/PTEN CRISPR engineered cell lines, indicating activation of downstream PI3K/AKT and MAPK signalling pathways in the osimertinib-resistant cells. Importantly, our in vitro experiments from multiple cell line models indicated that PIK3CA-mediated resistance to osimertinib could be partially reversed by co-treatment with AKT (capivasertib, AstraZeneca) and PI3K alpha (alpelisib, Novartis) inhibitors; PTEN-mediated resistance could be rescued by co-treatment with capivasertib and PI3K beta (AZD8186, AstraZeneca) inhibitors. We next examined the in vivo efficacy of osimertinib + AKT/PI3K inhibitors therapies in NSCLC xenograft models. Our in vivo analysis show that PIK3CA GOF CRISPR engineered cells displayed diminished response to osimertinib when implanted in mice; importantly, combination with AKT (capivasertib) or PI3K alpha (alpelisib) inhibitors enhanced response on treatment and delayed outgrowth after withdrawal treatment. In addition, we observed that combination treatment with capivasertib induced tumour stasis in a PIK3CA-E454K and two PTENloss PDX models.Altogether our in vitro and in vivo data provide evidence of PIK3CA mutants and PTEN loss-driven mechanisms of resistance to osimertinib and offer possible therapeutic combination strategies for those patients that develop resistance or experience a sub-optimal response to osimertinib through PIK3CA/PTEN mutations Citation Format: Ursula Grazini, Daniel J. O'Neill, Matthew Martin, Chintia Xu, Nicolas Floch, Paul D. Smith, Emanuela Cuomo. PIK3CA and PTEN mutations as drivers of osimertinib resistance in patients with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5353.</jats:p

Supplementary Methods, Figures 1-9 from ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors

Supplementary Methods, Figures 1-9 from ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors</jats:p

Clinical impact of subclonal EGFR T790M mutations in advanced-stage EGFR-mutant non-small-cell lung cancers

AbstractAdvanced non-small-cell lung cancer (NSCLC) patients with EGFR T790M-positive tumours benefit from osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). Here we show that the size of the EGFR T790M-positive clone impacts response to osimertinib. T790M subclonality, as assessed by a retrospective NGS analysis of 289 baseline plasma ctDNA samples from T790M‐positive advanced NSCLC patients from the AURA3 phase III trial, is associated with shorter progression-free survival (PFS), both in the osimertinib and the chemotherapy-treated patients. Both baseline and longitudinal ctDNA profiling indicate that the T790M subclonal tumours are enriched for PIK3CA alterations, which we demonstrate to confer resistance to osimertinib in vitro that can be partially reversed by PI3K pathway inhibitors. Overall, our results elucidate the impact of tumour heterogeneity on response to osimertinib in advanced stage NSCLC patients and could help define appropriate combination therapies in these patients.</jats:p