<p>Figure S1: Prevalence of N-terminal fusion partners and ROS1 exonic breakpoints; Figure S2: Activation of STAT3 and IRS1 by ROS1 fusions; Figure S3: Effects of MEK activation in ROS1 fusion oncoprotein-expressing cellular models; Figure S4: JAK/STAT pathway activation is unable to rescue ROS1 fusion-positive patient-derived cells from crizotinib sensitivity; Figure S5: Effects of MAPK pathway suppression by SHP2 inhibitor treatment in ROS1-fusion oncoprotein expressing patient-derived NSCLC cell lines; Figure S6: SHP2 interacts with phospho-SDC4-ROS1; Figure S7: ROS1 exonic breakpoint does not determine the fusion protein's ability to engage the MAPK pathway; Figure S8: Localization of ROS1 in patient-derived cell lines reveals differential subcellular localization of the different ROS1 fusion oncoproteins; Figure S9: Growth of positive and negative control NIH-3T3 cells in vivo; Figure S10: MAPK pathway activation is higher in SDC4-ROS1- and SLC34A2-ROS1-driven tumors</p>
Paf1C co-localizes with Pol II and influences gene expression by regulating transcription initiation, elongation and termination. Some crucial functions of Paf1C include promoting co-transcriptional histone modifications and recruiting termination factors. The mechanism of chromatin recruitment of Paf1C was obscure. We identified the importance of a conserved region within the Rtf1 subunit of Paf1C, termed the ORF association region (OAR), in chromatin-tethering of Paf1C. I found that the interaction of Paf1C with the transcription elongation factor Spt5 was mediated by the Rtf1 OAR and the Spt5 C-terminal region (CTR). Binding assays established the direct nature of the Rtf1-Spt5 interaction and the sufficiency of the Rtf1 OAR and the Spt5 CTR for this interaction. ChIP assays demonstrated the ability of the OAR to mimic the chromatin association pattern of Paf1C, independent of Paf1C but dependent on the Spt5 CTR and the Bur1 kinase. This suggests that the targeting of the OAR tethers Paf1C to chromatin. Collectively, these results provide a molecular mechanism for coupling Paf1C with the transcription machinery.
Additionally, I found that substitution of OAR residues predicted to be important for the human Rtf1 OAR-Spt5 CTR interaction in the OAR-CTR co-crystal impaired the chromatin association of Paf1C supporting the relevance of the co-crystal interactions. Furthermore, I showed that strains that are doubly mutated in the OAR and the Cdc73 C-domain exhibited cumulative reduction in Paf1C chromatin occupancy. Consistently, I showed that cells lacking both the OAR and the C-domain lose Paf1C-mediated histone modifications. This indicates that the Rtf1 OAR and the Cdc73 C-domain facilitate dual-attachment of Paf1C to chromatin.
My work has also provided better understanding of the function of the histone modification domain (HMD) of Rtf1. I found that overexpression of the HMD was essential for it to promote histone modifications. Additionally, I showed that the HMD is sufficient for the H2B K123 Ub, the mark upstream of the H3 K4 and H3 K79 methylation events, but the rest of Paf1C is required for the HMD to stimulate H3 K4 Me3 modification. Cumulatively, my findings provide additional insight into the regulation of histone modifications by the Rtf1 HMD.
Abstract Chromosomal rearrangements involving receptor tyrosine kinases (RTK) are a clinically relevant oncogenic mechanism in human cancers. These chimeric oncoproteins often contain the C-terminal kinase domain of the RTK joined in cis to various N-terminal, nonkinase fusion partners. The functional role of the N-terminal fusion partner in RTK fusion oncoproteins is poorly understood. Here, we show that distinct N-terminal fusion partners drive differential subcellular localization, which imparts distinct cell signaling and oncogenic properties of different, clinically relevant ROS1 RTK fusion oncoproteins. SDC4-ROS1 and SLC34A2-ROS1 fusion oncoproteins resided on endosomes and activated the MAPK pathway. CD74-ROS1 variants that localized instead to the endoplasmic reticulum (ER) showed compromised activation of MAPK. Forced relocalization of CD74-ROS1 from the ER to endosomes restored MAPK signaling. ROS1 fusion oncoproteins that better activate MAPK formed more aggressive tumors. Thus, differential subcellular localization controlled by the N-terminal fusion partner regulates the oncogenic mechanisms and output of certain RTK fusion oncoproteins. Significance: ROS1 fusion oncoproteins exhibit differential activation of MAPK signaling according to subcellular localization, with ROS1 fusions localized to endosomes, the strongest activators of MAPK signaling.
Abstract Despite recent advances in cancer treatment, lung cancer remains the leading cause of cancer mortality worldwide. Lung adenocarcinoma is the most prevalent subtype of lung cancer. Genomic profiling of lung adenocarcinomas has led to the identification of several targetable oncogenic drivers. Therapies targeting the oncogenic-driver pathway using various tyrosine kinase inhibitors (TKIs), are effective initially but responses are often transient and tumors eventually regrow due to drug resistance. Furthermore, drug resistance can arise via the selection of pre-existing resistant clones or via the de novo acquisition of mutations that are not present before therapy. We set out to understand the mechanism for the de novo acquisition of drug resistance mutations in oncogene-driven lung cancers. To do so, we investigated the gene expression changes that occur upon inhibition of oncogenic pathways. We found that oncoprotein targeted therapy induces adaptations favorable for APOBEC genome mutagenesis. Treatment with small molecule inhibitors against EGFR and ALK promoted transcriptional upregulation of members of APOBEC family of cytidine deaminases and downregulation of the uracil glycosylase UNG, the key protein needed for removal of APOBEC-induced DNA lesions. These changes in mRNA levels resulted in functional effects that can impact nuclear DNA by increasing nuclear APOBEC activity and reducing nuclear uracil excision capacity. Determination of changes in APOBEC mRNA levels and nuclear APOBEC activity over time and depletion studies identified APOBEC3B as a driver of both baseline and targeted therapy-induced nuclear APOBEC activity in pre-clinical lung cancer models. We found that APOBEC3B mediates genetic evolution and emergence of resistance during targeted therapy. We identified NF-kB pathway induction and c-Jun downregulation as key mediators of these treatment-induced molecular changes. Furthermore, we find an upregulation of APOBEC3B in lung cancer patients with progressive disease and a high proportion of APOBEC-associated mutations in patient tumors treated with targeted therapy. Some putative resistance mutations in patient tumors were also in the APOBEC-preferred context. Our study identifies a novel targeted therapy-induced evolutionary process involving an APOBEC DNA deaminase that could serve as an attractive co-target to elicit more durable treatment responses. Citation Format: Manasi K. Mayekar, Deborah Caswell, Natalie Vokes, Wei Wu, Caroline McCoach, Collin Blakely, Nuri Alpay Temiz, Daniel Lucas Kerr, Julia Rotow, Franziska Haderk, Lauren Cech, Beatrice Gini, Shigeki Nanjo, Lisa Tan, Johnny Yu, Carlos Gomez, Philippe Gui, Elizabeth Yu, Nicholas Thomas, Julian Downward, Reuben Harris, Eliezer Van Allen, Charles Swanton, Trever Bivona. APOBEC3B fuels evolution of resistance during targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB124.
Abstract Introduction: Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. Results: Using the multi-region TRACERx lung cancer study, we uncovered that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR driven lung cancer and identified subclonal enrichment of APOBEC mutational signatures. To model APOBEC mutagenesis in lung cancer, several novel EGFR mutant mouse models containing a human APOBEC3B transgene were generated. Using these models, it was uncovered that APOBEC3B expression is detrimental at tumor initiation when expressed continuously in a p53 wildtype background. This detrimental effect is likely due to elevated chromosomal instability, which was observed to increase significantly with APOBEC3B expression in an EGFR mutant TP53 deficient mouse model. Induction of subclonal expression of APOBEC3B in an EGFR mutant mouse model with tyrosine kinase inhibitor (TKI) therapy resulted in a significant increase in resistant tumor development. Significant downregulation of the base excision repair gene uracil-DNA glycosylase (UNG) was also observed in APOBEC3B expressing mice, which paralleled findings in patient tumors and cell lines treated with TKI therapy. Finally, a mouse mutational signature was identified in APOBEC3B expressing cell lines, reinforcing the idea that APOBEC driven mutagenesis contributes to TKI resistance. Conclusion: This study demonstrates a unique principle by which targeted therapy induces changes within tumors ideal for APOBEC driven tumor evolution, fueling therapy resistance. Citation Format: Manasi Mayekar, Deborah Caswell, Natalie Vokes, Emily K. Law, Wei Wu, William Hill, Eva Gronroos, Andrew Rowan, Maise Al Bakir, Clare Weeden, Caroline E. McCoach, Collin M. Blakely, Nuri Alpay Temiz, Ai Nagano, Daniel L. Kerr, Julia K. Rotow, Oriol Pich, Franziska Haderk, Michelle Dietzen, Carlos Martinez Ruiz, Bruna Almeida, Lauren Cech, Beatrice Gini, Joanna Przewrocka, Chris Moore, Miguel Murillo, Bjorn Bakker, Brandon Rule, Cameron Durfee, Shigeki Nanj, Lisa Tan, Lindsay K. Larson, Prokopios P. Argyris, William L. Brown, Johnny Yu, Carlos Gomez, Philippe Gui, Rachel I. Vogel, Elizabeth A. Yu, Nicholas J. Thomas, Subramanian Venkatesan, Sebastijan Hobor, Su Kit Chew, Nicholas McGranahan, Nnennaya Kanu, Eliezer M. Van Allen, Julian Downward, Reuben S. Harris, Trever Bivona, Charles Swanton. Targeted cancer therapy induces APOBEC fueling the evolution of drug resistance [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 2197.
<div>Abstract<p>Chromosomal rearrangements involving receptor tyrosine kinases (RTK) are a clinically relevant oncogenic mechanism in human cancers. These chimeric oncoproteins often contain the C-terminal kinase domain of the RTK joined <i>in cis</i> to various N-terminal, nonkinase fusion partners. The functional role of the N-terminal fusion partner in RTK fusion oncoproteins is poorly understood. Here, we show that distinct N-terminal fusion partners drive differential subcellular localization, which imparts distinct cell signaling and oncogenic properties of different, clinically relevant ROS1 RTK fusion oncoproteins. SDC4-ROS1 and SLC34A2-ROS1 fusion oncoproteins resided on endosomes and activated the MAPK pathway. CD74-ROS1 variants that localized instead to the endoplasmic reticulum (ER) showed compromised activation of MAPK. Forced relocalization of CD74-ROS1 from the ER to endosomes restored MAPK signaling. ROS1 fusion oncoproteins that better activate MAPK formed more aggressive tumors. Thus, differential subcellular localization controlled by the N-terminal fusion partner regulates the oncogenic mechanisms and output of certain RTK fusion oncoproteins.</p>Significance:<p>ROS1 fusion oncoproteins exhibit differential activation of MAPK signaling according to subcellular localization, with ROS1 fusions localized to endosomes, the strongest activators of MAPK signaling.</p></div>
Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae . Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6–Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of full-length Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.
Abstract Molecularly targeted cancer therapy has improved outcomes for cancer patients with targetable oncoproteins, such as mutant epidermal growth factor receptor ( EGFR ) in lung cancer. Yet, long-term patient survival remains limited because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations in the EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR . Here, we report the functional impact of inactivating genetic alteration of the mRNA splicing factor RBM10 that co-occur with mutant EGFR . RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of Bcl-xS-(pro-apoptotic)-to-Bcl-xL(anti-apoptotic) Bcl-x isoforms. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Co-inhibition of Bcl-xL and mutant EGFR overcame resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations, and on the impact of splicing factor deficiency in the modulation of sensitivity to targeted kinase inhibitor cancer therapy.
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