Increased chromatin accessibility drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer.

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations.

PIK3CA mutations occur in ∼8% of cancers, including ∼40% of HR-positive breast cancers, where the PI3K-alpha (PI3Kα)-selective inhibitor alpelisib is FDA approved in combination with fulvestrant. Although prior studies have identified resistance mechanisms, such as PTEN loss, clinically acquired resistance to PI3Kα inhibitors remains poorly understood. Through serial liquid biopsies and rapid autopsies in 39 patients with advanced breast cancer developing acquired resistance to PI3Kα inhibitors, we observe that 50% of patients acquire genomic alterations within the PI3K pathway, including PTEN loss and activating AKT1 mutations. Notably, although secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Kα inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket. Some mutations had differential effects on PI3Kα-selective versus pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Kα-inhibitor RLY-2608. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. SIGNIFICANCE: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

WNT5a Signaling through ROR2 Activates the Hippo Pathway to Suppress YAP1 Activity and Tumor Growth.

Noncanonical Wnt signaling by WNT5a has oncogenic and tumor suppressive activities, but downstream pathways mediating these specific effects remain to be fully established. In a subset of prostate cancer organoid culture and xenograft models, inhibition of Wnt synthesis stimulated growth, whereas WNT5a or a WNT5a mimetic peptide (Foxy5) markedly suppressed tumor growth. WNT5a caused a ROR2-dependent decrease in YAP1 activity, which was associated with increased phosphorylation of MST1/2, LATS1, MOB1, and YAP1, indicating Hippo pathway activation. Deletion of MST1/2 abrogated the WNT5a response. WNT5a similarly activated Hippo in ROR2-expressing melanoma cells, whereas WNT5a in ROR2-negative cells suppressed Hippo. This suppression was associated with increased inhibitory phosphorylation of NF2/Merlin that was not observed in ROR2-expressing cells. WNT5a also increased mRNA encoding Hippo pathway components including MST1 and MST2 and was positively correlated with these components in prostate cancer clinical datasets. Conversely, ROR2 and WNT5a expression was stimulated by YAP1, and correlated with increased YAP1 activity in clinical datasets, revealing a WNT5a/ROR2 negative feedback loop to modulate YAP1 activity. Together these findings identify Hippo pathway activation as a mechanism that mediates the tumor suppressive effects of WNT5a and indicate that expression of ROR2 may be a predictive biomarker for responsiveness to WNT5a-mimetic drugs. SIGNIFICANCE: WNT5a signaling through ROR2 activates the Hippo pathway to downregulate YAP1/TAZ activity and suppress tumor growth, identifying ROR2 as a potential biomarker to identify patients that could benefit from WNT5a-related agents.

Plk1 Inhibitors and Abiraterone Synergistically Disrupt Mitosis and Kill Cancer Cells of Disparate Origin Independently of Androgen Receptor Signaling.

Abiraterone is a standard treatment for metastatic castrate-resistant prostate cancer (mCRPC) that slows disease progression by abrogating androgen synthesis and antagonizing the androgen receptor (AR). Here we report that inhibitors of the mitotic regulator polo-like kinase-1 (Plk1), including the clinically active third-generation Plk1 inhibitor onvansertib, synergizes with abiraterone in vitro and in vivo to kill a subset of cancer cells from a wide variety of tumor types in an androgen-independent manner. Gene-expression analysis identified an AR-independent synergy-specific gene set signature upregulated upon abiraterone treatment that is dominated by pathways related to mitosis and the mitotic spindle. Abiraterone treatment alone caused defects in mitotic spindle orientation, failure of complete chromosome condensation, and improper cell division independently of its effects on AR signaling. These effects, although mild following abiraterone monotherapy, resulted in profound sensitization to the antimitotic effects of Plk1 inhibition, leading to spindle assembly checkpoint-dependent mitotic cancer cell death and entosis. In a murine patient-derived xenograft model of abiraterone-resistant metastatic castration-resistant prostate cancer (mCRPC), combined onvansertib and abiraterone resulted in enhanced mitotic arrest and dramatic inhibition of tumor cell growth compared with either agent alone. Overall, this work establishes a mechanistic basis for the phase II clinical trial (NCT03414034) testing combined onvansertib and abiraterone in mCRPC patients and indicates this combination may have broad utility for cancer treatment. SIGNIFICANCE: Abiraterone treatment induces mitotic defects that sensitize cancer cells to Plk1 inhibition, revealing an AR-independent mechanism for this synergistic combination that is applicable to a variety of cancer types.

Clinical Activity and Safety of Cabozantinib for Brain Metastases in Patients With Renal Cell Carcinoma.

IMPORTANCE: Patients with brain metastases from renal cell carcinoma (RCC) have been underrepresented in clinical trials, and effective systemic therapy is lacking. Cabozantinib shows robust clinical activity in metastatic RCC, but its effect on brain metastases remains unclear. OBJECTIVE: To assess the clinical activity and toxic effects of cabozantinib to treat brain metastases in patients with metastatic RCC. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study included patients with metastatic RCC and brain metastases treated in 15 international institutions (US, Belgium, France, and Spain) between January 2014 and October 2020. Cohort A comprised patients with progressing brain metastases without concomitant brain-directed local therapy, and cohort B comprised patients with stable or progressing brain metastases concomitantly treated by brain-directed local therapy. EXPOSURES: Receipt of cabozantinib monotherapy at any line of treatment. MAIN OUTCOMES AND MEASURES: Intracranial radiological response rate by modified Response Evaluation Criteria in Solid Tumors, version 1.1, and toxic effects of cabozantinib. RESULTS: Of the 88 patients with brain metastases from RCC included in the study, 33 (38%) were in cohort A and 55 (62%) were in cohort B; the majority of patients were men (n = 69; 78%), and the median age at cabozantinib initiation was 61 years (range, 34-81 years). Median follow-up was 17 months (range, 2-74 months). The intracranial response rate was 55% (95% CI, 36%-73%) and 47% (95% CI, 33%-61%) in cohorts A and B, respectively. In cohort A, the extracranial response rate was 48% (95% CI, 31%-66%), median time to treatment failure was 8.9 months (95% CI, 5.9-12.3 months), and median overall survival was 15 months (95% CI, 9.0-30.0 months). In cohort B, the extracranial response rate was 38% (95% CI, 25%-52%), time to treatment failure was 9.7 months (95% CI, 6.0-13.2 months), and median overall survival was 16 months (95% CI, 12.0-21.9 months). Cabozantinib was well tolerated, with no unexpected toxic effects or neurological adverse events reported. No treatment-related deaths were observed. CONCLUSIONS AND RELEVANCE: In this cohort study, cabozantinib showed considerable intracranial activity and an acceptable safety profile in patients with RCC and brain metastases. Support of prospective studies evaluating the efficacy of cabozantinib for brain metastases in patients with RCC is critical.

Targeting the Intrinsic Apoptosis Pathway: A Window of Opportunity for Prostate Cancer.

Despite major improvements in the management of advanced prostate cancer over the last 20 years, the disease remains invariably fatal, and new effective therapies are required. The development of novel hormonal agents and taxane chemotherapy has improved outcomes, although primary and acquired resistance remains problematic. Inducing cancer cell death via apoptosis has long been an attractive goal in the treatment of cancer. Apoptosis, a form of regulated cell death, is a highly controlled process, split into two main pathways (intrinsic and extrinsic), and is stimulated by a multitude of factors, including cellular and genotoxic stress. Numerous therapeutic strategies targeting the intrinsic apoptosis pathway are in clinical development, and BH3 mimetics have shown promising efficacy for hematological malignancies. Utilizing these agents for solid malignancies has proved more challenging, though efforts are ongoing. Molecular characterization and the development of predictive biomarkers is likely to be critical for patient selection, by identifying tumors with a vulnerability in the intrinsic apoptosis pathway. This review provides an up-to-date overview of cell death and apoptosis, specifically focusing on the intrinsic pathway. It summarizes the latest approaches for targeting the intrinsic apoptosis pathway with BH3 mimetics and discusses how these strategies may be leveraged to treat prostate cancer.

Covid-19 positivity affects outcome of cardiac surgical patients.

INTRODUCTION: In this report we describe the clinical presentation, laboratory findings and outcomes of four patients that were referred for urgent cardiothoracic intervention and tested positive for COVID-19. METHODS: The St. Elizabeth's Medical Center Institutional Review Board exempted the study from review (waived review). In each case, verbal informed consent was obtained by the study participant or health care proxy. RESULTS: The majority of the patients undergoing surgery had low Society of Thoracic Surgeons score and uneventful operating time. The mortality was very high and driven primarily by the viral syndrome. Laboratory markers that have been associated with disease severity in the general population were also prognostic in our population. CONCLUSION: Our study shows that these patients have very high mortality, whereas prevention and preoperative screening is required in preventing nosocomial spreading of the disease.

Small cell transformation of non-small cell lung cancer on immune checkpoint inhibitors: uncommon or under-recognized?

BACKGROUND: Histological transformation of oncogene-driven lung adenocarcinoma to small cell lung cancer (SCLC) following treatment with tyrosine kinase inhibitors (TKIs) is a well-described phenomenon. Whether a similar transformation may drive acquired resistance to immune checkpoint inhibitors (ICPIs) in non-SCLC (NSCLC) is uncertain. Hence, tissue biopsies are not universally recommended at progression of NSCLC on ICPIs, unlike TKIs. CASE PRESENTATION: We report a case of a woman in her mid-60s with a 35 pack-years tobacco history and stage IV squamous cell lung carcinoma with no targetable genomic alterations, whose disease progressed within 4 months of first line carboplatin/gemcitabine therapy. Her treatment was switched to second line nivolumab monotherapy which resulted in sustained partial response lasting 21 months. She subsequently developed rapid, bulky progression of mediastinal disease. Biopsy showed transformation to SCLC. Comparison of genomic profiling results from the initial NSCLC diagnosis and SCLC transformation revealed near-identical tumor profiles. Her disease responded to next line carboplatin/etoposide, though lasting for only 10 months. She died 14 months after detection of neuroendocrine transformation of her NSCLC. SYSTEMATIC REVIEW: We performed a systematic review of the literature to identify similar cases of NSCLC-to-small cell transformation on ICPIs. Nine patients, including our index case, were identified, with seven (77.8%) on nivolumab and two (22.2%) on pembrolizumab monotherapy. Median survival time since small cell transformation was 13.0 months (95% CI 2.0 to 16.0). Using our patient case as a framework, we further discuss the lack of consensus criteria to distinguish small cell transformation from de novo metachronous SCLC. CONCLUSIONS: Histological transformation to SCLC is a potential mechanism of acquired resistance to ICPIs in NSCLC. Repeat tissue biopsies should be considered at the time of progression, similar to oncogene-directed therapies. Prospective larger studies are warranted to further characterize NSCLC-to-small cell transformation on ICPIs using molecular fingerprinting with paired tumor genomic profiles, evaluation of neuroendocrine features at baseline and consideration of initial response.

MARCH5 mediates NOXA-dependent MCL1 degradation driven by kinase inhibitors and integrated stress response activation.

MCL1 has critical antiapoptotic functions and its levels are tightly regulated by ubiquitylation and degradation, but mechanisms that drive this degradation, particularly in solid tumors, remain to be established. We show here in prostate cancer cells that increased NOXA, mediated by kinase inhibitor activation of an integrated stress response, drives the degradation of MCL1, and identify the mitochondria-associated ubiquitin ligase MARCH5 as the primary mediator of this NOXA-dependent MCL1 degradation. Therapies that enhance MARCH5-mediated MCL1 degradation markedly enhance apoptosis in response to a BH3 mimetic agent targeting BCLXL, which may provide for a broadly effective therapy in solid tumors. Conversely, increased MCL1 in response to MARCH5 loss does not strongly sensitize to BH3 mimetic drugs targeting MCL1, but instead also sensitizes to BCLXL inhibition, revealing a codependence between MARCH5 and MCL1 that may also be exploited in tumors with MARCH5 genomic loss.

Combining Immune Checkpoint and VEGFR Inhibition in Favorable Risk and Elderly Patients With Metastatic Renal Cell Carcinoma.

BACKGROUND: Immune checkpoint inhibitors and vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors are the most commonly used medications in metastatic renal cell carcinoma (mRCC). Recently, large clinical trials have shown favorable outcomes in patients treated with combined immune checkpoint plus VEGFR inhibition compared with VEGFR inhibition alone. However, the benefit among favorable risk (based on International Metastatic Renal Cell Carcinoma Database Consortium score) and elderly (age > 65 years) patients was not clear, leading to a discrepancy between United States Food and Drug Administration and European Association of Urology recommendations. MATERIALS AND METHODS: We searched available literature for phase III randomized clinical trials evaluating the efficacy of combining immunotherapy plus VEGF/VEGFR inhibitors versus standard of care in patients with previously untreated mRCC. Combinations that were included in United States Food and Drug Administration recommendations or European Association of Urology guidelines were used for analysis. We performed a meta-analysis with a random effects model to evaluate the efficacy of immunotherapy-VEGFR inhibitor combinations compared with sunitinib in favorable risk and elderly patients. RESULTS: Our analysis demonstrated that progression-free survival (PFS) was significantly prolonged with combination therapy compared with sunitinib in patients with age > 65 years (hazard ratio, 0.66; 95% confidence interval, 0.52-0.84; P = .001). The PFS in favorable risk disease was improved with combination therapy compared with sunitinib, but the difference was not statistically significant (hazard ratio, 0.68; 95% confidence interval, 0.46-1.01; P = .055). CONCLUSION: Our meta-analysis strengthens the trend of beneficial effect in prolonging PFS in both subgroups compared with each trial alone, indicating that favorable risk and elderly patients with mRCC likely benefit from combining programmed cell death 1 or programmed cell death-ligand 1 and VEGFR inhibition.

EGFR-A763_Y764insFQEA Is a Unique Exon 20 Insertion Mutation That Displays Sensitivity to Approved and In-Development Lung Cancer EGFR Tyrosine Kinase Inhibitors.

INTRODUCTION: The EGFR-A763_Y764insFQEA is a unique exon 20 insertion mutation (~5% to 6% of exon 20 insertions), which, at the structural and enzyme kinetic level, more closely resembles EGFR tyrosine kinase inhibitor (TKI)- sensitizing mutants, such as EGFR exon 19 indels and L858R. A limited number of preclinical models and clinical reports have studied the response of this mutant to EGFR TKIs. METHODS: We used models of EGFR-A763_Y764insFQEA and more typical EGFR exon 20 insertion mutations to probe representative first- (gefitinib, erlotinib), second- (afatinib), third-generation (osimertinib), and in-development EGFR exon 20-specific (poziotinib, mobocertinib [TAK-788]) TKIs. We also compiled outcomes of EGFR-A763_Y764insFQEA-mutated lung cancers treated with EGFR TKIs. RESULTS: Cells driven by EGFR-A763_Y764insFQEA were consistently sensitive to EGFR TKIs (as opposed to those driven by typical EGFR exon 20 insertions [A767_V769dupASV, D770_N771insSVD and H773_V774insH]), which were only inhibited by in-development EGFR TKIs at doses below those affecting wild-type EGFR. Most case instances (62.5% [95% confidence interval: 39%-86%], n = 16) with lung cancers harboring EGFR-A763_Y764insFQEA responded to clinically available EGFR TKIs (including osimertinib) and to in-development EGFR exon 20-specific TKIs (including mobocertinib) with prolonged periods of progression-free survival in some cases. Median overall survival for EGFR TKI-treated cases was 22 months (95% confidence interval: 16-25). Mechanisms of acquired TKI resistance of this mutant remain underreported, but do seem to align with those of common mutations. CONCLUSIONS: To our knowledge, this is the largest report to confirm that the EGFR-A763_Y764insFQEA mutation is sensitive to clinically available first-, second-, third-generation, and in-development EGFR TKIs.

Unusual Presentation of Hemophagocytic Lymphohistiocytosis in a Kidney Transplant Patient.

We are presenting a case of a middle-aged woman with history of remote kidney transplantation who had multiple admissions for septic shock-like picture, recurrent fever, and hypotension. Her shock manifestation would resolve after stress dose steroid administration and less than 24 hours of vasopressor administration. Initially, extensive workup was performed without revealing etiology. Eventually, a bone marrow biopsy was carried out leading to the diagnosis of hemophagocytic lymphohistiocytosis, most likely related to recent cytomegalovirus infection.

Circulating inflammation signature predicts overall survival and relapse-free survival in metastatic colorectal cancer.

BACKGROUND: Metastatic colorectal cancer (mCRC) is a highly heterogeneous disease from a clinical, molecular, and immunological perspective. Current predictive models rely primarily in tissue based genetic analysis, which not always correlate with inflammatory response. Here we evaluated the role of a circulating inflammatory signature as a prognostic marker in mCRC. METHODS: Two hundred eleven newly diagnosed patients with mCRC were enrolled in the study. One hundred twenty-one patients had unresectable metastases, whereas ninety patients had potentially resectable liver metastases at presentation. Analysis of miR-21, IL-6, and IL-8 in the plasma of peripheral blood was performed at baseline. Patients with high circulating levels of ≥2 of the three inflammation markers (miR-21, IL-6, and IL-8) were considered to have the "Inflammation phenotype-positive CISIG". RESULTS: Positive CISIG was found in 39/90 (43%) and 50/121 (45%) patients in the resectable and unresectable cohort, respectively. In the resectable population the median relapse-free survival was 18.4 compared to 31.4 months (p = 0.001 HR 2.09, 95% CI 1.2-3.67) for positive vs. negative CISIG. In contrast, the individual components were not significant. In the same population the median overall survival was 46.2 compared to 66.0 months (p = 0.0003, HR 2.57, 95% CI 1.26-5.27) for positive vs. negative CISIG, but not significant for the individual components. In the unresectable population, the median overall survival was 13.5 compared to 25.0 months (p = 0.0008, HR 2.49, 95% CI 1.46-4.22) for positive vs. negative CISIG. IL-6 was independently prognostic with overall survival of 16.2 compared to 27.0 months (p = 0.004, HR 1.96, 95% CI 1.24-3.11) for high vs. low IL-6, but not the other components. Using a Cox regression model, we demonstrated that CISIG is an independent predictive marker of survival in patients with unresectable disease (HR 1.8, 95% CI 1.2, 2.8, p < 0.01). CONCLUSION: In two different cohorts, we demonstrated that CISIG is a strong prognostic factor of relapse-free and overall survival of patients with mCRC. Based on these data, analysis of circulating inflammatory signaling can be complimentary to traditional molecular testing.

Modeling of Patient-Derived Xenografts in Colorectal Cancer.

Developing realistic preclinical models using clinical samples that mirror complex tumor biology and behavior are vital to advancing cancer research. While cell line cultures have been helpful in generating preclinical data, the genetic divergence between these and corresponding primary tumors has limited clinical translation. Conversely, patient-derived xenografts (PDX) in colorectal cancer are highly representative of the genetic and phenotypic heterogeneity in the original tumor. Coupled with high-throughput analyses and bioinformatics, these PDXs represent robust preclinical tools for biomarkers, therapeutic target, and drug discovery. Successful PDX engraftment is hypothesized to be related to a series of anecdotal variables namely, tissue source, cancer stage, tumor grade, acquisition strategy, time to implantation, exposure to prior systemic therapy, and genomic heterogeneity of tumors. Although these factors at large can influence practices and patterns related to xenotransplantation, their relative significance in determining the success of establishing PDXs is uncertain. Accordingly, we systematically examined the predictive ability of these factors in establishing PDXs using 90 colorectal cancer patient specimens that were subcutaneously implanted into immunodeficient mice. Fifty (56%) PDXs were successfully established. Multivariate analyses showed tissue acquisition strategy [surgery 72.0% (95% confidence interval (CI): 58.2-82.6) vs. biopsy 35% (95% CI: 22.1%-50.6%)] to be the key determinant for successful PDX engraftment. These findings contrast with current empiricism in generating PDXs and can serve to simplify or liberalize PDX modeling protocols. Better understanding the relative impact of these factors on efficiency of PDX formation will allow for pervasive integration of these models in care of colorectal cancer patients. Mol Cancer Ther; 16(7); 1435-42. ©2017 AACR.