Acetylation of MLH1 by CBP increases cellular DNA mismatch repair activity.

The DNA mismatch repair (MMR) proteins recognize and repair DNA base pair mismatches and insertions/deletions of DNA that have occurred during DNA replication. Additionally, they are involved in regulation of the DNA damage response, including cell cycle checkpoints and apoptosis. Therefore, regulation of these proteins is essential for maintaining genomic integrity. It has been recognized that post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, are being used as an important means to regulate the functions and stability of MMR proteins. Here, we report that a histone acetyltransferase CREB binding protein (CBP) interacts with and acetylates MLH1, a component of the MutLα complex (MLH1-PMS2). Moreover, CBP stabilizes MLH1 by preventing it from degradation via the ubiquitin-proteasome degradation pathway. Consistently, acetylation induced by a pan-histone deacetylase inhibitor, Trichostatin A, promotes the assembly between the MutSα (MSH2-MSH6) and MutLα complexes. Furthermore, overexpression of CBP enhances MMR activities in cells. Overall, our results suggest a novel role of CBP in prolonging MLH1 stability and enhancing MutSα-MutLα complex formation, leading to increased cellular MMR activity.

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model.

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform.

ATR inhibition overcomes platinum tolerance associated with ERCC1- and p53-deficiency by inducing replication catastrophe.

ERCC1/XPF is a heterodimeric DNA endonuclease critical for repair of certain chemotherapeutic agents. We recently identified that ERCC1- and p53-deficient lung cancer cells are tolerant to platinum-based chemotherapy. ATR inhibition synergistically re-stored platinum sensitivity to platinum tolerant ERCC1-deficient cells. Mechanistically we show this effect is reliant upon several functions of ATR including replication fork protection and altered cell cycle checkpoints. Utilizing an inhibitor of replication protein A (RPA), we further demonstrate that replication fork protection and RPA availability are critical for platinum-based drug tolerance. Dual treatment led to increased formation of DNA double strand breaks and was associated with chromosome pulverization. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATR inhibition may also enhance response to immunotherapy in ERCC1-deficient tumors. In vivo studies demonstrate a significant effect on tumor growth delay with combination therapy compared with single agent treatment. Results of this study have led to the identification of a feasible therapeutic strategy combining ATR inhibition with platinum and potentially immune checkpoint blockade inhibitors to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.

The Mechanisms of Zinc Action as a Potent Anti-Viral Agent: The Clinical Therapeutic Implication in COVID-19.

The pandemic of COVID-19 was caused by a novel coronavirus termed as SARS-CoV2 and is still ongoing with high morbidity and mortality rates in the whole world. The pathogenesis of COVID-19 is highly linked with over-active immune and inflammatory responses, leading to activated cytokine storm, which contribute to ARDS with worsen outcome. Currently, there is no effective therapeutic drug for the treatment of COVID-19. Zinc is known to act as an immune modulator, which plays an important role in immune defense system. Recently, zinc has been widely considered as an anti-inflammatory and anti-oxidant agent. Accumulating numbers of studies have revealed that zinc plays an important role in antiviral immunity in several viral infections. Several early clinical trials clearly indicate that zinc treatment remarkably decreased the severity of the upper respiratory infection of rhinovirus in humans. Currently, zinc has been used for the therapeutic intervention of COVID-19 in many different clinical trials. Several clinical studies reveal that zinc treatment using a combination of HCQ and zinc pronouncedly reduced symptom score and the rates of hospital admission and mortality in COVID-19 patients. These data support that zinc might act as an anti-viral agent in the addition to its anti-inflammatory and anti-oxidant properties for the adjuvant therapeutic intervention of COVID-19.

A Randomized, Open-Label Phase II Study Evaluating Emibetuzumab Plus Erlotinib and Emibetuzumab Monotherapy in MET Immunohistochemistry Positive NSCLC Patients with Acquired Resistance to Erlotinib.

INTRODUCTION: The hepatocyte growth factor receptor MET represents a resistance mechanism to epidermal growth factor receptor (EGFR) inhibition in EGFR mutant (mt) non-small cell lung cancer (NSCLC). This Phase 2 study tested whether acquired resistance to erlotinib in MET protein positive NSCLC patients enriched for EGFRmt can be overcome by emibetuzumab plus erlotinib. PATIENT AND METHODS: Patients with Stage IV NSCLC with acquired resistance to erlotinib and MET diagnostic (+) (≥ 10% of cells expressing MET at ≥ 2+ IHC staining intensity at any time) were randomized (3:1) to receive emibetuzumab 750 mg every 2 weeks with or without erlotinib 150 mg once daily. The primary objective was to evaluate the overall response rate (ORR) relative to historic control, with a co-primary objective of ORR in patients with MET expression in ≥ 60% of cells ≥ 2+ (MET ≥ 60%). RESULTS: One hundred and eleven MET+ patients received emibetuzumab plus erlotinib (N = 83) or emibetuzumab monotherapy (N = 28). 89 of 111 MET+ samples were post-erlotinib. ORR was 3.0% for emibetuzumab plus erlotinib (95% CI: 0.4, 10.5) and 4.3% for emibetuzumab (95% CI: 0.1, 21.9), in patients with post-erlotinib progression biopsies available (n = 89). Similar results were observed in patients with MET ≥ 60% expression (n = 74). Disease control rate and progression-free survival were higher for emibetuzumab plus erlotinib (50%/3.3 months) than for emibetuzumab (26%/1.6 months). No unexpected safety signals emerged. Partial responses were observed in patients with and without EGFRmt or MET amplification. EGFR sensitizing mutations were identified retrospectively in 84.2% of those with available tissue (85/101). CONCLUSION: Acquired resistance to erlotinib in MET diagnostic (+) patients was not reversed by emibetuzumab plus erlotinib or emibetuzumab monotherapy, although a subset of patients obtained clinical benefit.

The Effects of HER2 Alterations in EGFR Mutant Non-small Cell Lung Cancer.

BACKGROUND: HER2 alteration (mutation and/or amplification) is associated with poor survival in NSCLC and can mediate resistance to EGFR tyrosine kinase inhibitors. METHODS: We retrospectively analyzed de-identified molecular information from 12,946 NSCLC samples that underwent next-generation sequencing (NGS) with Caris Life Sciences. The objectives were to determine the prevalence and type of HER2 alterations with and without EGFR as a co-mutation. Insurance claims were utilized to obtain outcomes data. RESULTS: Three hundred and twenty-one patients (2.5%) had HER2 alteration: mutation in 197 patients and amplification in 134. Median age was 65 years and 62% were female. A total of 84% were adenocarcinoma. HER2 exon 20 insertion was most common (69%). A total of 1551 (12%) patients had EGFR mutations. Among samples with EGFR mutations, 24 (1.5%) had concurrent HER2 alteration (8 with HER2 mutation and 16 with amplification). Among 8 patients who had both EGFR and HER2 mutations, 3 had EGFR exon 19 deletions and exon 8 HER2 mutation (S310F). One-third of the patients (7/21) with HER2 extracellular domain (ECD) mutation had co-occurring EGFR mutations. All 7 were S310. Patients with concurrent EGFR mutation and HER2 amplification had longer median time on treatment with EGFR TKI(s) than those with EGFR mutation without HER2 amplification (HR 2.284, P =.004). CONCLUSION: A minority of NSCLC samples with EGFR mutations had HER2 alterations. In patients with both mutations, exon 21 mutations for EGFR and exon 8 mutations for HER2 were common. It will be critical to continue to accumulate valuable clinical data for further real-world outcomes analysis.

Implications of the USP10-HDAC6 axis in lung cancer - A path to precision medicine.

Lung cancer is the leading cause of cancer death among both men and women in the United States. Because lung cancer is genetically heterogeneous, tailored therapy alone or in combination with chemotherapy would increase patient overall survival as compared with the one-size-fits-all chemotherapy. TP53-mutant lung cancer accounts for more than half of all lung cancer cases and is oftentimes more aggressive and resistant to chemotherapy. Directly targeting mutant p53 has not yet been successful, so identification of novel therapy targets and biomarkers in the TP53-mutant lung cancer is urgently needed to increase the overall survival in this subgroup. Deubiquitinating enzymes (DUBs) regulate a vast majority of proteins (DUBs' substrates) via removal of ubiquitin moieties or ubiquitin chains from these proteins, thereby altering the stability and/or functions of these substrates. In this review, we will focus on a DUB, referred to as ubiquitin-specific peptidase 10 (USP10) whose substrates include both oncogenic proteins and tumor suppressors. Therefore, targeting USP10 in cancer is highly context-dependent. Here, we will discuss USP10's functions in cancer by examining its various known substrates. In particular, we will elaborate our recent findings in the oncogenic role of USP10 in the TP53-mutant subgroup of lung cancer, focusing on USP10's function in the DNA damage response (DDR) via histone deacetylase 6 (HDAC6). Overall, these findings support the notion that targeting USP10 in the TP53-mutant subgroup of NSCLC would sensitize patients to cisplatin-based chemotherapy. Generating potent and specific clinically relevant USP10 inhibitors would benefit the TP53-mutant subgroup of NSCLC patients.

Impact of XPO1 mutations on survival outcomes in metastatic non-small cell lung cancer (NSCLC).

BACKGROUND: Nuclear protein transport is essential in guiding the traffic of important proteins and RNAs between the nucleus and cytoplasm. Export of proteins from the nucleus is mostly regulated by Exportin 1 (XPO1). In cancer, XPO1 is almost universally hyperactive and can promote the export of important tumor suppressors to the cytoplasm. Currently, there are no studies evaluating XPO1 amplifications and mutations in NSCLC and the impact on outcomes. METHODS: Tumor samples were analyzed using next-generation sequencing (NGS) (NextSeq, 592 Genes), immunohistochemistry (IHC), and whole transcriptome sequencing (WTS, NovaSeq) (Caris Life Sciences, Phoenix, AZ). Survival was extracted from insurance claims data and calculated from time of tissue collection to last contact using Kaplan-Meier estimate. RESULTS: Among 18,218 NSCLC tumors sequenced, 26 harbored XPO1 mutations and 24 had amplifications. XPO1 mutant tumors were more likely to have high TMB (79% vs. 52%, p = 0.007) and less likely to have high PD-L1 (32% vs. 68%, p = 0.03). KRAS co-mutations were seen in 19% (n = 5) and EGFR co-mutations were rare (n = 2). Among the 17,449 NSCLC tumors with clinical data, there were 24 XPO1 mutant. Comparison of survival between XPO1 mutant and WT showed a negative association with a hazard ratio (HR) of 1.932 (95% CI: 1.144-3.264 p = 0.012). XPO1 amplification was not associated with survival. CONCLUSIONS: XPO1 pathogenic mutations were associated with a poor survival in NSCLC. Although XPO1 mutations are rare in NSCLC, further studies to assess its associations with treatment responses are warranted.

Role of novel cancer gene SLITRK3 to activate NTRK3 in squamous cell lung cancer.

The development of targeted therapies that inhibit cancer-driving oncogenes has improved outcomes of patients diagnosed with lung adenocarcinoma (LUAD). In contrast, patients diagnosed with lung squamous cell carcinoma (LUSC) suffer worse survival outcomes and lack effective targeted treatment options. Identification of molecular drivers of LUSC to support development of targeted treatments is urgently needed. Addressing this need, the current report introduces the novel cancer gene SLIT- and NTRK-like family member 3 (SLITRK3) and its role in activating the neurotrophic receptor tyrosine kinase 3 (NTRK3) in LUSC cells. Multiple genome-wide data sets from patient samples were produced by us or downloaded from public databases to analyze tumor gene copy number aberrations, mRNA expression and associated survival outcomes. An accompanying mechanistic study employed LUSC cell lines and multiple methods, including in situ immunofluorescence, sphere-formation assay, and fluorescence-activated cell sorting analysis of the CD133-positive cell fraction. Altogether, the results indicate that gene amplification and consequent high expression of SLITRK3 in LUSC is associated with worse outcomes and induces SLITRK3-dependent activation of NTRK3 to promote a cancer stem cell phenotype that is inhibited by existing NTRK-targeted inhibitors. Based on a recent literature search, this is the first report of a mechanistic role for SLITRK3 in cancer.

HDAC6 Regulates Radiosensitivity of Non-Small Cell Lung Cancer by Promoting Degradation of Chk1.

We have previously discovered that HDAC6 regulates the DNA damage response (DDR) via modulating the homeostasis of a DNA mismatch repair protein, MSH2, through HDAC6's ubiquitin E3 ligase activity. Here, we have reported HDAC6's second potential E3 ligase substrate, a critical cell cycle checkpoint protein, Chk1. We have found that HDAC6 and Chk1 directly interact, and that HDAC6 ubiquitinates Chk1 in vivo and in vitro. Specifically, HDAC6 interacts with Chk1 via the DAC1 domain, which contains its ubiquitin E3 ligase activity. During the cell cycle, Chk1 protein levels fluctuate, peaking at the G2 phase, subsequently resolving via the ubiquitin-proteasome pathway, and thereby allowing cells to progress to the M phase. However, in HDAC6 knockdown non-small cell lung cancer (NSCLC) cells, Chk1 is constitutively active and fails to resolve post-ionizing radiation (IR), and this enhanced Chk1 activity leads to preferential G2 arrest in HDAC6 knockdown cells accompanied by a reduction in colony formation capacity and viability. Depletion or pharmacological inhibition of Chk1 in HDAC6 knockdown cells reverses this radiosensitive phenotype, suggesting that the radiosensitivity of HDAC6 knockdown cells is dependent on increased Chk1 kinase activity. Overall, our results highlight a novel mechanism of Chk1 regulation at the post-translational level, and a possible strategy for sensitizing NSCLC to radiation via inhibiting HDAC6's E3 ligase activity.

The USP10-HDAC6 axis confers cisplatin resistance in non-small cell lung cancer lacking wild-type p53.

Ubiquitin-specific peptidase 10 (USP10) stabilizes both tumor suppressors and oncogenes in a context-dependent manner. However, the nature of USP10's role in non-small cell lung cancer (NSCLC) remains unclear. By analyzing The Cancer Genome Atlas (TCGA) database, we have shown that high levels of USP10 are associated with poor overall survival in NSCLC with mutant p53, but not with wild-type p53. Consistently, genetic depletion or pharmacological inhibition of USP10 dramatically reduces the growth of lung cancer xenografts lacking wild-type p53 and sensitizes them to cisplatin. Mechanistically, USP10 interacts with, deubiquitinates, and stabilizes oncogenic protein histone deacetylase 6 (HDAC6). Furthermore, reintroducing either USP10 or HDAC6 into a USP10-knockdown NSCLC H1299 cell line with null-p53 renders cisplatin resistance. This result suggests the existence of a "USP10-HDAC6-cisplatin resistance" axis. Clinically, we have found a positive correlation between USP10 and HDAC6 expression in a cohort of NSCLC patient samples. Moreover, we have shown that high levels of USP10 mRNA correlate with poor overall survival in a cohort of advanced NSCLC patients who received platinum-based chemotherapy. Overall, our studies suggest that USP10 could be a potential biomarker for predicting patient response to platinum, and that targeting USP10 could sensitize lung cancer patients lacking wild-type p53 to platinum-based therapy.

Physical activity and quality of life in African American cancer survivors: The Detroit Research on Cancer Survivors study.

BACKGROUND: The benefit of regular exercise in improving cancer outcomes is well established. The American Cancer Society (ACS) released a recommendation that cancer survivors should engage in at least 150 minutes of moderate to vigorous physical activity (PA) per week; however, few report meeting this recommendation. This study examined the patterns and correlates of meeting ACS PA recommendations in the Detroit Research on Cancer Survivors (ROCS) cohort of African American cancer survivors. METHODS: Detroit ROCS participants completed baseline and yearly follow-up surveys to update their health and health behaviors, including PA. This study examined participation in PA by select characteristics and reported health-related quality of life (HRQOL) as measured with the Functional Assessment of Cancer Therapy and Patient-Reported Outcomes Measurement Information System instruments. RESULTS: Among the first 1500 ROCS participants, 60% reported participating in regular PA, with 24% reporting ≥150 min/wk. Although there were no differences by sex, prostate cancer survivors were the most likely to report participating in regular PA, whereas lung cancer survivors were the least likely (P = .022). Survivors who reported participating in regular PA reported higher HRQOL (P < .001) and lower depression (P = .040). CONCLUSIONS: Just 24% of African American cancer survivors reported meeting the ACS guidelines for PA at the baseline, but it was encouraging to see increases in activity over time. Because of the established benefits of regular exercise observed in this study and others, identifying and reducing barriers to regular PA among African American cancer survivors are critical for improving outcomes and minimizing disparities.

Profiling the Mutational Landscape in Known Driver Genes and Novel Genes in African American Non-Small Cell Lung Cancer Patients.

PURPOSE: Identifying novel driver genes and mutations in African American non-small cell lung cancer (NSCLC) cases can inform targeted therapy and improve outcomes for this traditionally underrepresented population. EXPERIMENTAL DESIGN: Tumor DNA, RNA, and germline DNA were collected from African American NSCLC patients who participated in research conducted at the Karmanos Cancer Institute (KCI) in Detroit, Michigan. Known mutations were ascertained through the Sequenom LungCarta panel of 214 mutations in 26 genes, RET/ROS1 fusions, amplification of FGFR1, and expression of ALK. Paired tumor and normal DNA was whole-exome sequenced for a subset of cases without known driver mutations. RESULTS: Of the 193 tumors tested, 77 known driver mutations were identified in 66 patients (34.2%). Sixty-seven of the 127 patients without a known driver mutation were sequenced. In 54 of these patients, 50 nonsynonymous mutations were predicted to have damaging effects among the 26 panel genes, 47 of which are not found in The Cancer Genome Atlas NSCLC white or African American samples. Analyzing the whole-exome sequence data using MutSig2CV identified a total of 88 genes significantly mutated at FDR q < 0.1. Only 5 of these genes were previously reported as oncogenic. CONCLUSIONS: These findings suggest that broader mutation profiling including both known and novel driver genes in African Americans with NSCLC will identify additional mutations that may be useful in treatment decision-making.

HDAC6 regulates DNA damage response via deacetylating MLH1.

MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage.

Identification and Characterization of Synthetic Viability with ERCC1 Deficiency in Response to Interstrand Crosslinks in Lung Cancer.

PURPOSE: ERCC1/XPF is a DNA endonuclease with variable expression in primary tumor specimens, and has been investigated as a predictive biomarker for efficacy of platinum-based chemotherapy. The failure of clinical trials utilizing ERCC1 expression to predict response to platinum-based chemotherapy suggests additional mechanisms underlying the basic biology of ERCC1 in the response to interstrand crosslinks (ICLs) remain unknown. We aimed to characterize a panel of ERCC1 knockout (Δ) cell lines, where we identified a synthetic viable phenotype in response to ICLs with ERCC1 deficiency. EXPERIMENTAL DESIGN: We utilized the CRISPR-Cas9 system to create a panel of ERCC1Δ lung cancer cell lines which we characterized. RESULTS: We observe that loss of ERCC1 hypersensitizes cells to cisplatin when wild-type (WT) p53 is retained, whereas there is only modest sensitivity in cell lines that are p53mutant/null. In addition, when p53 is disrupted by CRISPR-Cas9 (p53*) in ERCC1Δ/p53WT cells, there is reduced apoptosis and increased viability after platinum treatment. These results were recapitulated in 2 patient data sets utilizing p53 mutation analysis and ERCC1 expression to assess overall survival. We also show that kinetics of ICL-repair (ICL-R) differ between ERCC1Δ/p53WT and ERCC1Δ/p53* cells. Finally, we provide evidence that cisplatin tolerance in the context of ERCC1 deficiency relies on DNA-PKcs and BRCA1 function. CONCLUSIONS: Our findings implicate p53 as a potential confounding variable in clinical assessments of ERCC1 as a platinum biomarker via promoting an environment in which error-prone mechanisms of ICL-R may be able to partially compensate for loss of ERCC1.See related commentary by Friboulet et al., p. 2369.

Randomized-controlled phase II trial of salvage chemotherapy after immunization with a TP53-transfected dendritic cell-based vaccine (Ad.p53-DC) in patients with recurrent small cell lung cancer.

Small cell lung cancer TP53 mutations lead to expression of tumor antigens that elicits specific cytotoxic T-cell immune responses. In this phase II study, dendritic cells transfected with wild-type TP53 (vaccine) were administered to patients with extensive-stage small cell lung cancer after chemotherapy. Patients were randomized 1:1:1 to arm A (observation), arm B (vaccine alone), or arm C (vaccine plus all-trans-retinoic acid). Vaccine was administered every 2 weeks (3 times), and all patients were to receive paclitaxel at progression. Our primary endpoint was overall response rate (ORR) to paclitaxel. The study was not designed to detect overall response rate differences between arms. Of 69 patients enrolled (performance status 0/1, median age 62 years), 55 were treated in stage 1 (18 in arm A, 20 in arm B, and 17 in arm C) and 14 in stage 2 (arm C only), per 2-stage Simon Minimax design. The vaccine was safe, with mostly grade 1/2 toxicities, although 1 arm-B patient experienced grade 3 fatigue and 8 arm-C patients experienced grade 3 toxicities. Positive immune responses were obtained in 20% of arm B (95% confidence interval [CI], 5.3-48.6) and 43.3% of arm C (95% CI 23.9-65.1). The ORRs to the second-line chemotherapy (including paclitaxel) were 15.4% (95% CI 2.7-46.3), 16.7% (95% CI 2.9-49.1), and 23.8% (95% CI 9.1-47.5) for arms A, B, and C, with no survival differences between arms. Although our vaccine failed to improve ORRs to the second-line chemotherapy, its safety profile and therapeutic immune potential remain. Combinations with the other immunotherapeutic agents are reasonable options.

Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor.

Dexamethasone (Dex), co-administered to lung adenocarcinoma patients with pemetrexed chemotherapy, protects against pemetrexed cytotoxicity by inducing reversible G1 arrest, reflected by the effect of Dex on FLT-PET images of patient tumors. However, perioperative Dex treatment increases survival but the mechanism is unknown. In cells with glucocorticoid receptor-α (GR) expression corresponding to higher clinical tumor levels, Dex-induced growth arrest was followed by marked cell expansion, beta-galactosidase expression and Ki67 negativity, despite variable p53 and K-RAS status. Dex induced a transient early surge in p21Cip1. However, a progressive, irreversible loss of clonogenic growth, whose time of onset was dependent on GR level and Dex dose, was independent of p21Cip1and caused by gradual accumulation of p27Kip1 due to transcriptional activation of p27Kip1 by Dex. This effect was independent of canonical pathways of senescence or p27Kip1 regulation. The in vitro observations were reflected by growth suppression and P27Kip1 induction in GR-overexpressing tumor xenografts compared with isogenic low-GR tumors. Extended Dex treatment induces irreversible cell cycle blockade and a senescence phenotype through chronic activation of the p27Kip1 gene in GR overexpressing lung tumor cell populations and hence could improve outcome of surgery/pemetrexed chemotherapy and sensitize tumors to immunotherapy.

Prognostic modeling of the immune-centric transcriptome reveals interleukin signaling candidates contributing to differential patient outcomes.

Immunotherapy is a promising advancement in the treatment of non-small-cell lung carcinoma (NSCLC), although much of how lung tumors interact with the immune system in the natural course of disease remains unknown. We investigated the impact of the expression of immune-centric genes and pathways in tumors on patient survival to reveal novel candidates for immunotherapeutic research. Tumor transcriptomes and detailed clinical characteristics were obtained from patients with NSCLC who were participants of either the Inflammation, Health and Lung Epidemiology (INHALE) (discovery, N = 280) or The Cancer Genome Atlas (TCGA) Lung (replication, N = 1026) studies. Expressions of 2253 genes derived from 48 major immune pathways were assessed for association with patient prognosis using a multivariable Cox model and pathway effects were assessed with an in-house implementation of the Gene Set Enrichment Analysis (GSEA) algorithm. Prognosis-guided gene and pathway analysis of immune-centric expression in tumors revealed significant survival enrichments across both cohorts. The 'Interleukin Signaling' pathway, containing 430 genes, was found to be statistically and significantly enriched with prognostic signal in both the INHALE (P = 0.008) and TCGA (P = 0.039) datasets. Subsequent leading-edge analysis identified a subset of genes (N = 23) shared between both cohorts, driving the pathway enrichment. Cumulative expression of this leading-edge gene signature was a strong predictor of patient survival [discovery: hazard ratio (HR) = 1.59, P = 3.0 × 10-8; replication: HR = 1.29, P = 7.4 × 10-7]. These data demonstrate the impact of immune-centric expression on patient outcomes in NSCLC. Furthermore, prognostic gene effects were localized to discrete immune pathways, of which Interleukin Signaling had the greatest impact on overall survival and the subset of genes driving these effects have promise for future therapeutic intervention.

A CARP-1 functional mimetic compound is synergistic with BRAF-targeting in non-small cell lung cancers.

Non-small cell lung cancers (NSCLC) account for 85% of all lung cancers, and the epidermal growth factor receptor (EGFR) is highly expressed or activated in many NSCLC that permit use of EGFR tyrosine kinase inhibitors (TKIs) as frontline therapies. Resistance to EGFR TKIs eventually develops that necessitates development of improved and effective therapeutics. CARP-1/CCAR1 is an effector of apoptosis by Doxorubicin, Etoposide, or Gefitinib, while CARP-1 functional mimetic (CFM) compounds bind with CARP-1, and stimulate CARP-1 expression and apoptosis. To test whether CFMs would inhibit TKI-resistant NSCLCs, we first generated and characterized TKI-resistant NSCLC cells. The GI 50 dose of Erlotinib for parental and Erlotinib-resistant HCC827 cells was ∼0.1 µM and ≥15 µM, respectively. While Rociletinib or Ocimertinib inhibited the parental H1975 cells with GI 50 doses of ≤0.18 µM, the Ocimertinib-resistant pools of H1975 cells had a GI50 dose of ∼12 µM. The GI50 dose for Rociletinib-resistant H1975 sublines ranged from 4.5-8.0 µM. CFM-4 and its novel analog CFM-4.16 attenuated growth of the parental and TKI-resistant NSCLC cells. CFMs activated p38/JNKs, inhibited oncogenic cMet and Akt kinases, while CARP-1 depletion blocked NSCLC cell growth inhibition by CFM-4.16 or Erlotinib. CFM-4.16 was synergistic with B-Raf-targeting in NSCLC, triple-negative breast cancer, and renal cancer cells. A nano-lipid formulation (NLF) of CFM-4.16 in combination with Sorafenib elicited a superior growth inhibition of xenografted tumors derived from Rociletinib-resistant H1975 NSCLC cells in part by stimulating CARP-1 and apoptosis. These findings support therapeutic potential of CFM-4.16 together with B-Raf targeting in treatment of TKI-resistant NSCLCs.

RUMI is a novel negative prognostic marker and therapeutic target in non-small-cell lung cancer.

Recent comprehensive next-generation genome and transcriptome analyses in lung cancer patients, several clinical observations, and compelling evidence from mouse models of lung cancer have uncovered a critical role for Notch signaling in the initiation and progression of non-small-cell lung cancer (NSCLC). Notably, Rumi is a "protein O-glucosyltransferase" that regulates Notch signaling through O-glucosylation of Notch receptors, and is the only enzymatic regulator whose activity is required for both ligand-dependent and ligand-independent activation of Notch. We have conducted a detailed study on RUMI's involvement in NSCLC development and progression, and have further explored the therapeutic potential of its targeting in NSCLC. We have determined that Rumi is highly expressed in the alveolar and bronchiolar epithelia, including club cells and alveolar type II cells. Remarkably, RUMI maps to the region of chromosome 3q that corresponds to the major signature of neoplastic transformation in NSCLC, and is markedly amplified and overexpressed in NSCLC tumors. Notably, RUMI expression levels are predictive of poor prognosis and survival in NSCLC patients. Our data indicates that RUMI modulates Notch activity in NSCLC cells, and that its silencing dramatically decreases cell proliferation, migration, and survival. RUMI downregulation causes severe cell cycle S-phase arrest, increases genome instability, and induces late apoptotic-nonapoptotic cell death. Our studies demonstrate that RUMI is a novel negative prognostic factor with significant therapeutic potential in NSCLC, which embodies particular relevance especially when considering that, while current Notch inhibitory strategies target only ligand-dependent Notch activation, a large number of NSCLCs are driven by ligand-independent Notch activity.