Abstract: Although a subset of genetic loci have been associated with gastric cancer (GC) risk, the underlying mechanisms are largely unknown. We aimed to identify new susceptibility genes and elucidate their mechanisms in GC development. Design We conducted a meta-analysis of four genome-wide association studies (GWASs) encompassing 3771 cases and 5426 controls. After targeted sequencing and functional annotation, we performed in vitro and in vivo experiments to confirm the functions of genetic variants and candidate genes. Moreover, we selected 33 promising variants for two-stage replication in 7035 cases and 8323 controls from other five studies. Results The meta-analysis of GWASs identified three loci at 1q22, 5p13.1 and 10q23.33 associated with GC risk at p 〈 5×10 − 8  and replicated seven known loci at p 〈 0.05. At 5p13.1, the risk rs59133000[C] allele enhanced the binding affinity of NF-κB1 (nuclear factor kappa B subunit 1) to the promoter of PRKAA1 , resulting in a reduced promoter activity and lower expression. The knockout of PRKAA1 promoted both GC cell proliferation and xenograft tumour growth in nude mice. At 10q23.33, the rs3781266[C] and rs3740365[T] risk alleles in complete linkage disequilibrium disrupted and created, respectively, the binding motifs of POU2F1 and PAX3, resulting in an increased enhancer activity and expression of NOC3L , while the NOC3L knockdown suppressed GC cell growth. Moreover, two new loci at 3q11.2 (OR=1.21, p=4.56×10 − 9 ) and 4q28.1 (OR=1.14, p=3.33×10 − 11 ) were associated with GC risk. Conclusion We identified 12 loci to be associated with GC risk in Chinese populations and deciphered the mechanisms of PRKAA1 at 5p13.1 and NOC3L at 10q23.33 in gastric tumourigenesis.

Abstract: What's new? There is an urgent need for more effective conditioning therapies for refractory and relapsed lymphomas. In this Phase II study, the authors found that sensitizing lymphoma cells with a histone deacetylase inhibitor (HDACi) enhances the cytotoxicity of CGB chemotherapy, effectively preventing relapse after stem‐cell transplant, and improves long‐term overall survival.

Abstract: BACKGROUND: Pharmacological targeting of driver alterations in cancer has resulted in many clinical successes but is limited by concurrent or novel genomic alterations. One potential explanation for this heterogeneity is the presence of additional genomic alterations which modify the degree of dependence on the targeted driver mutation. Metastatic prostate cancer (mPCa) serves as a relevant example, where the molecular target is the androgen receptor (AR) which functions as a lineage survival factor of luminal prostate epithelial cells. Next generation AR targeted therapies such as abiraterone, enzalutamide and apalutamide have significantly improved the survival of men with mPCa and achieved exciting clinical success. However, resistance to these therapies with disease progression is unfortunately inevitable, with intrinsic resistance noted in around 30% patients and acquired resistance in most patients. Therefore, there is an unmet need to understand the mechanism of therapy resistance to AR targeted therapies and identify novel therapeutic approach to prevent or reverse resistance. Previously, we have revealed that the deactivation of two genes, TP53 and RB1, confers AR targeted therapy resistance through a novel mechanism by which tumor cells acquire lineage plasticity and transit to a multi-lineage, progenitor-like state no longer dependent on AR. This lineage plasticity and resistance is enabled by the activation of SOX2 and is completely reversible by knocking down SOX2. This observation not only adds clarity to the mechanism of resistance, but also suggests that appropriate clinical interventions of lineage plasticity may be a potential avenue to overcome resistance. However, there is only 10% mPCa patients carrying homozygous deletions in both TP53 and RB1 loci, thus additional genomic alterations may be responsible for the resistance in other patients. METHODS: To gain functional insight into the genes impacted by the copy number alterations in mPCa, we screened 4234 short hairpin RNAs (shRNAs) targeting 730 genes often deleted in human prostate cancer (annotated from a survey of six prostate cancer genomic datasets) for hairpins that confer in vivo resistance to the antiandrogen enzalutamide, in a well credentialed enzalutamide-sensitive xenograft model LNCaP/AR. More than 350 resistant tumors emerged by 16 weeks of xenografting and the genomic DNA of these tumors were extracted and sequenced to determine the enrichment of specific shRNAs compared to the starting material. A classic probabilistic model RIGER-E was used to determine the significance of enrichment of each hairpins/genes. RESULTS: The chromodomain helicase DNA-binding protein 1 (CHD1) emerged as a top candidate, a finding supported by patient data showing that CHD1 expression is inversely correlated with clinical benefit from AR targeted therapy enzalutamide. CRISPR based depletion of CHD1 confers significant resistance to enzalutamide both in vitro and in vivo, supported by similar results from multiple human prostate cancer cell line models. To our surprise, we observed sustained inhibition of the canonical AR target genes, indicating that CHD1 loss might activate transcriptional programs that relieve prostate tumor cells from their dependence on AR by reprogramming away from their luminal lineage, as we have observed in the setting of combined loss of RB1 and TP53. Indeed, CHD1 loss led to global changes in open and closed chromatin, indicative of an altered chromatin state, with associated changes in gene expression. Integrative analysis of ATAC-seq and RNA-seq changes identified 22 transcription factors as candidate drivers of enzalutamide resistance. CRISPR deletion of four of these (NR3C1, BRN2, NR2F1, TBX2) restored in vitro enzalutamide sensitivity in CHD1 deleted cells. Independently derived, enzalutamide-resistant, CHD1-deleted subclones expressed elevated levels of 1 or more of these 4 transcription factors. This pattern suggests a state of chromatin plasticity and enhanced heterogeneity, initiated by CHD1 loss, which enables upregulation of distinct sets of genes in response to selective pressure. This concept is further supported by RNA-seq data from a mCRPC patients cohort, in which we examined the co-association of CHD1 levels with each of these four TFs across 212 tumors. Unsupervised clustering analysis of just these five genes identified five distinct clusters, four of which display relatively higher expression of either CHD1 or one or two of these four resistance TFs. Interestingly, we observed altered expression of many canonical lineage specific genes in the same panel of CHD1-deleted, enzalutamide resistant xenografts that displayed heterogenous upregulation of the four TFs, including consistent downregulation of luminal genes and upregulation of genes specify epithelial to mesenchymal transition (EMT). Furthermore, these upregulation of 4 resistance TFs, as well as the observed lineage switchs, are both rapid and reversible, suggesting a status of plasticity. Collectively, these results indicate that CHD1 loss establishes an altered chromatin landscape which, in the face of stresses such as antiandrogen therapy, enables resistant subclones to emerge through activation of alternative, non-luminal lineage programs that reduce dependence on AR. CONCLUSIONS: We demonstrated that loss of the chromodomain gene CHD1, a commonly deleted prostate cancer gene (in 15~20% patients), through global effects on chromatin, establishes a state of plasticity that accelerates the development of AR targeted therapy resistance through heterogeneous activation of downstream effectors, which mediated the transition away from luminal lineage identity and AR dependency. This model provides the first demonstration that early genomic lesions of critical epigenetic modulator promotes prostate cancer heterogeneity and lineage plasticity, consequently leading to the resistance to AR targeted therapy. Therefore, appropriate clinical intervention of these heterogenous resistance driver TFs, as well as the chromatin dysregulation, may be potential therapeutic avenues to prevent or reverse AR targeted resistance. Citation Format: Zeda Zhang, Chuanli Zhou, Xiaoling Li, Spencer Barnes, Su Deng, Elizabeth Hoover, Chi-Chao Chen, Young Sun Lee, Choushi Wang, Carla Tirado, Lauren Metang, Nickolas Johnson, John Wongvipat, Kristina Navrazhina, Zhen Cao, Wassim Abida, Amaia Lujambio, Sheng Li, Vankat Malladi, Charles Sawyers, Ping Mu. CHD1-loss confers AR targeted therapy resistance via promoting cancer heterogeneity and lineage plasticity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG06.

Abstract: Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A1, A2a, A2b, A3) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A2 receptor agonists and was blocked by selective A2a and A2b receptor antagonists, indicating that adenosine was mediating its effect through A2 receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A2 receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.

Abstract: Targeted therapies for driver oncogenes have transformed the management of many cancers but the magnitude and duration of response remains variable. One potential explanation for the various response is the presence of additional genomic alterations which modify the degree of dependence on the targeted driver mutation. Metastatic castration resistant prostate cancer (mCRPC) serves as an example, where the target is the androgen receptor (AR). Compared to primary disease, mCRPC is characterized by extensive heterogeneity at both genomic and transcriptional levels, including genomic copy number alterations (CNAs), which are presumed to contribute to the resistance to AR targeted therapies. To gain functional insight into the genes impacted by the copy number alterations in mCRPC, we screened 730 genes often deleted in prostate cancer for CNAs that confer in vivo resistance and identified the chromodomain helicase DNA-binding protein 1 (CHD1) as a top candidate modifying resistance, a finding supported by patient data showing that CHD1 expression is inversely correlated with clinical benefit from therapy. Depletion of CHD1 in multiple human prostate cancer cell lines confers significant resistance to enzalutamide both in vitro and in vivo. Furthermore, we observed global changes in open and closed chromatin after the depletion of CHD1, indicative of an altered chromatin state, with associated changes in gene expression. Integrative analysis of ATAC-seq and RNA-seq, combining with CRISPR-based screen, identified four heterogenous resistance drivers (GR, BRN2, NR2F1, TBX2), which are elevated in different independently derived, enzalutamide-resistant, CHD1-deleted subclones. Importantly, significantly increased transcriptional heterogeneity was observed in these CHD1-depleted resistant tumors and in the tumor samples from a large mCRPC patients’ cohort. Finally, GR inhibition restored the enzalutamide sensitiivty in resistant tumors with elevated GR signaling. These results suggest CHD1-loss, through global effects on chromatin, establishes a state of plasticity that accelerates the development of AR targeted therapy resistance through activation of heterogeneous downstream effectors, which mediated the transition away from luminal lineage identity and AR dependency. This model not only provides an innovative explanation for the significantly increased transcriptional heterogeneity observed in mCRPC patients, but also suggests that proper clinical interventions targeting these heterogenous resistance drivers may be a novel avenue to prevent or even reverse resistance towards AR targeted therapies. Citation Format: Zeda Zhang, Chuanli Zhou, Xiaoling Li, Charles Sawyers, Ping Mu. CHD1-loss promotes tumor heterogeneity and therapy resistance in prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-117.