A Randomized Clinical Trial of Precision Prevention Materials Incorporating MC1R Genetic Risk to Improve Skin Cancer Prevention Activities Among Hispanics

Purpose: Skin cancer incidence is increasing among Hispanics, who experience worse outcomes than non-Hispanic Whites. Precision prevention incorporating genetic testing for (melanocortin-1 receptor) MC1R, a skin cancer susceptibility marker, may improve prevention behavior. Experimental Design: Hispanic participants (n = 920) from Tampa, FL and Ponce, PR, were block-randomized within MC1R higher- and average-risk groups to precision prevention or generic prevention arms. We collected baseline information on demographics, family history of cancer, phenotypic characteristics, health literacy, health numeracy, and psychosocial measures. Participants reported weekday and weekend sun exposure (in hours), number of sunburns, frequency of five sun protection behaviors, intentional outdoor and indoor tanning, and skin examinations at baseline, 3 months, and 9 months. Participants also reported these outcomes for their eldest child ≤10 years old. Results: Among MC1R higher-risk participants, precision prevention increased sunscreen use (OR = 1.74, P = 0.03) and receipt of a clinical skin exam (OR = 6.51, P = 0.0006); and it decreased weekday sun exposure hours (β = −0.94, P = 0.005) and improved sun protection behaviors (β = 0.93, P = 0.02) in their children. There were no significant intervention effects among MC1R average-risk participants. The intervention did not elevate participant cancer worry. We also identified moderators of the intervention effect among both average- and higher-risk participants. Conclusions: Receipt of MC1R precision prevention materials improved some skin cancer prevention behaviors among higher-risk participants and their children and did not result in reduced prevention activities among average-risk participants. Despite these encouraging findings, levels of sun protection behaviors remained suboptimal among participants, warranting more awareness and prevention campaigns targeted to Hispanics Significance: Our results support a precision public health approach to reducing skin cancers among Hispanics, an underserved population in precision medicine, and may additionally improve preventive behaviors among their children.

An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

Phase I Study of High-Dose l-Methylfolate in Combination with Temozolomide and Bevacizumab in Recurrent IDH Wild-Type High-Grade Glioma

Isocitrate dehydrogenase (IDH) mutations in low-grade gliomas (LGG) result in improved survival and DNA hypermethylation compared with IDH wild-type LGGs. IDH-mutant LGGs become hypomethylated during progression. It is uncertain whether methylation changes occur during IDH wild-type GBM progression and whether the methylome can be reprogrammed. This phase I study evaluated the safety, tolerability, efficacy, and methylome changes after l-methylfolate (LMF) treatment, in combination with temozolomide and bevacizumab in patients with recurrent high-grade glioma. Fourteen patients total, 13 with GBM, one with anaplastic astrocytoma, all IDH wild-type were enrolled in the study. All patients received LMF at either 15, 30, 60, or 90 mg daily plus temozolomide (75 mg/m2 5 days per month) and bevacizumab (10 mg/kg every two weeks).No MTD was identified. LMF-treated patients had median overall survival of 9.5 months [95% confidence interval (CI), 9.1–35.4] comparable with bevacizumab historical control 8.6 months (95% CI, 6.8–10.8). Six patients treated with LMF survived more than 650 days. Across all treatment doses, the most adverse events were diarrhea (7%, 1 patient, grade 2), reflux (7%, 1 patient, grade 2), and dysgeusia (7%, 1 patient, grade 2). In the six brains donated at death, there was a 25% increase in DNA methylated CpGs compared with the paired initial tumor. LMF in combination with temozolomide and bevacizumab was well tolerated in patients with recurrent IDH wild-type high-grade glioma. This small study did not establish a superior efficacy with addition of LMF compared with standard bevacizumab therapy; however, this study did show methylome reprogramming in high-grade glioma. Significance: Glioblastoma (GBM) is a primary brain tumor with a poor prognosis. Therapies to date have failed to improve survival. LGGs, with IDH mutations, have increased global DNA methylation and increased survival compared with GBMs. GBMs lack this mutation and have less DNA methylation. Here we show that the DNA methylome can be modified in GBM with LMF. Such treatment might be useful in methylome priming prior to immunotherapy.

Influence of Germline BRCA Genotype on the Survival of Patients with Triple-Negative Breast Cancer

The presence of BRCA pathogenic variants (PV) in triple-negative breast cancer (TNBC) is associated with a distinctive genomic profile that makes the tumor particularly susceptible to DNA-damaging treatments. However, patients with BRCA PVs can develop treatment resistance through the appearance of reversion mutations and restored BRCA expression. As copy-number variants (CNV) could be less susceptible to reversion mutations than point mutations, we hypothesize that carriers of BRCA CNVs may have improved survival after treatment compared with carriers of other BRCA PVs or BRCA wild-type. Women diagnosed with stage I–III TNBC at ≤50 years at a cancer center in Mexico City were screened for BRCA PVs using a recurrent PV assay (HISPANEL; 77% sensitivity). Recurrence-free survival (RFS) and overall survival (OS) were compared according to the mutational status. Among 180 women, 17 (9%) were carriers of BRCA1 ex9–12del CNVs and 26 (14%) of other BRCA PVs. RFS at ten years for the whole cohort was 79.2% [95% confidence interval (CI), 72.3–84.6], with no significant differences according to mutational status. 10-year OS for the entire cohort was 85.3% (95% CI, 78.7–90.0), with BRCA CNV carriers demonstrating numerically superior OS rates other PV carriers and noncarriers (100% vs. 78.6% and 84.7%; log-rank P = 0.037 and P = 0.051, respectively). This study suggests that BRCA1 ex9–12del CNV carriers with TNBC may have a better OS, and supports the hypothesis that the genotype of BRCA PVs may influence survival by limiting treatment resistance mediated by reversion mutations among CNV carriers. Significance: Large CNV BRCA carriers in a cohort of young Mexican patients with TNBC had superior OS rates than carriers of other BRCA pathogenic variants (i.e., small indels or point mutations). We hypothesize that this is due to the resistance of CNVs to reversion mutations mediating resistance to therapy. If validated, these findings have important prognostic and clinical treatment implications for BRCA-associated breast cancers.

Sex- and Mutation-Specific p53 Gain-of-Function Activity in Gliomagenesis

In cancer, missense mutations in the DNA-binding domain of TP53 are common. They abrogate canonical p53 activity and frequently confer gain-of-oncogenic function (GOF) through localization of transcriptionally active mutant p53 to noncanonical genes. We found that several recurring p53 mutations exhibit a sex difference in frequency in patients with glioblastoma (GBM). In vitro and in vivo analysis of three mutations, p53R172H, p53Y202C, and p53Y217C, revealed unique interactions between cellular sex and p53 GOF mutations that determined each mutation's ability to transform male versus female primary mouse astrocytes. These phenotypic differences were correlated with sex- and p53 mutation–specific patterns of genomic localization to the transcriptional start sites of upregulated genes belonging to core cancer pathways. The promoter regions of these genes exhibited a sex difference in enrichment for different transcription factor DNA-binding motifs. Together, our data establish a novel mechanism for sex-specific mutant p53 GOF activity in GBM with implications for all cancer. Significance: Sex differences in cancer, including glioblastoma, have been observed in both incidence and outcome. We reveal that TP53, the most commonly mutated gene in cancer, contributes to sex differences through differential GOF activity. This discovery has critical implications for our understanding of p53 mutations and the importance of sex as a biological variable.

The ADAM17-directed Inhibitory Antibody MEDI3622 Antagonizes Radiotherapy-induced VEGF Release and Sensitizes Non–Small Cell Lung Cancer for Radiotherapy

The cellular response to ionizing radiation (IR) depends on tumor cell and microenvironmental factors. Here, we investigated the role of IR-induced ADAM17 matrix metalloproteinase activity for the intercellular communication between tumor cells and the tumor vasculature in non–small cell lung cancer (NSCLC) tumor models. Factors shed by ADAM17 from NSCLC tumor cells (A549, H358) and relevant for endothelial cell migration were investigated using transwell migration assays, ELISA, and flow cytometry. Tumor angiogenesis–related endpoints were analyzed with the chorio-allantoic membrane assay and in murine NSCLC tumor models. Efficacy-oriented experiments were performed in a murine orthotopic NSCLC tumor model using irradiation with an image-guided small-animal radiotherapy platform alone and in combination with the novel ADAM17-directed antibody MEDI3622. In vitro, VEGF was identified as the major factor responsible for IR-induced and ADAM17-dependent endothelial cell migration toward attracting tumor cells. IR strongly enhanced tumor cell–associated ADAM17 activity, released VEGF in an ADAM17-dependent manner, and thereby coordinated the communication between tumor and endothelial cells. In vivo, tumor growth and microvessel size and density were strongly decreased in response to the combined treatment modality of IR and MEDI3622 but not by either treatment modality alone and thus suggest that the supra-additive effect of the combined treatment modality is in part due to abrogation of the ADAM17-mediated IR-induced protective effect on the tumor vasculature. Furthermore, we demonstrate that the novel ADAM17-inhibitory antibody MEDI3622 potently improves the radiotherapy response of NSCLC. Significance: The tumor response to radiotherapy is influenced by several factors of the tumor microenvironment. We demonstrate that inhibition of the sheddase ADAM17 by the novel antibody MEDI3622 reduces IR-induced VEGF release from tumor cells relevant for endothelial cell migration and vasculature protection, thereby enhancing radiotherapy treatment outcome of NSCLC.

Tumor Suppressor PLK2 May Serve as a Biomarker in Triple-Negative Breast Cancer for Improved Response to PLK1 Therapeutics

Polo-like kinase (PLK) family members play important roles in cell-cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, frequent loss of chromosome 5q11–35, which includes PLK2, is observed in basal-like breast cancer. In this study, we found that PLK2 was tumor suppressive in breast cancer, preferentially in basal-like and triple-negative breast cancer (TNBC) subtypes. Knockdown of PLK1 rescued phenotypes induced by PLK2 loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase through the kinase but not the polo-box domains of PLK2, suggesting PLK2 functioned at least partially through the interaction with PLK1. Furthermore, an improved treatment response was seen in both Plk2-deleted/low mouse preclinical and patient-derived xenograft (PDX) TNBC models using the PLK1 inhibitor volasertib alone or in combination with carboplatin. Reexpression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. In summary, this study delineates the effects of chromosome 5q loss in TNBC that includes PLK2, the relationship between PLK2 and PLK1, and how this may render PLK2-deleted/low tumors more sensitive to PLK1 inhibition in combination with chemotherapy. Significance: The tumor-suppressive role of PLK2, and its relationship with oncogene PLK1, provide a mechanistic rationalization to use PLK1 inhibitors in combination with chemotherapy to treat PLK2-low/deleted tumors. TNBC, and other cancers with low PLK2 expression, are such candidates to leverage precision medicine to identify patients who might benefit from treatment with these inhibitors.

Combinatorial Natural Killer Cell–based Immunotherapy Approaches Selectively Target Chordoma Cancer Stem Cells

Chordoma is a rare tumor derived from notochord remnants that has a propensity to recur and metastasize despite conventional multimodal treatment. Cancer stem cells (CSC) are implicated in chordoma's resistant and recurrent behavior; thus, strategies that target CSCs are of particular interest. Using in vitro cytotoxicity models, we demonstrated that anti-programmed death ligand 1 (anti–PD-L1; N-601) and anti-EGFR (cetuximab) antibodies enhanced lysis of chordoma cells by healthy donor and chordoma patient NK cells through antibody-dependent cellular cytotoxicity (ADCC). Treatment of NK cells with an IL15 superagonist complex (N-803) increased their cytotoxicity against chordoma cells, which was further enhanced by treatment with N-601 and/or cetuximab. PD-L1–targeted chimeric antigen receptor NK cells (PD-L1 t-haNKs) were also effective against chordoma cells. CSCs were preferentially vulnerable to NK-cell killing in the presence of N-601 and N-803. Flow cytometric analysis of a chordoma CSC population showed that CSCs expressed significantly more NK-activating ligand B7-H6 and PD-L1 than non-CSCs, thus explaining a potential mechanism of selective targeting. These data suggest that chordoma may be effectively targeted by combinatorial NK cell–mediated immunotherapeutic approaches and that the efficacy of these approaches in chordoma and other CSC-driven tumor types should be investigated further in clinical studies. Significance: Combinatory immunotherapy using NK-mediated approaches demonstrates robust antitumor activity in preclinical models of chordoma and selectively targets chordoma CSCs.

In Silico Epitope Prediction Analyses Highlight the Potential for Distracting Antigen Immunodominance with Allogeneic Cancer Vaccines

Allogeneic cancer vaccines are designed to induce antitumor immune responses with the goal of impacting tumor growth. Typical allogeneic cancer vaccines are produced by expansion of established cancer cell lines, transfection with vectors encoding immunostimulatory cytokines, and lethal irradiation. More than 100 clinical trials have investigated the clinical benefit of allogeneic cancer vaccines in various cancer types. Results show limited therapeutic benefit in clinical trials and currently there are no FDA-approved allogeneic cancer vaccines. We used recently developed bioinformatics tools including the pVACseq suite of software tools to analyze DNA/RNA-sequencing data from the The Cancer Genome Atlas to examine the repertoire of antigens presented by a typical allogeneic cancer vaccine, and to simulate allogeneic cancer vaccine clinical trials. Specifically, for each simulated clinical trial, we modeled the repertoire of antigens presented by allogeneic cancer vaccines consisting of three hypothetical cancer cell lines to 30 patients with the same cancer type. Simulations were repeated ten times for each cancer type. Each tumor sample in the vaccine and the vaccine recipient was subjected to human leukocyte antigen (HLA) typing, differential expression analyses for tumor-associated antigens (TAA), germline variant calling, and neoantigen prediction. These analyses provided a robust, quantitative comparison between potentially beneficial TAAs and neoantigens versus distracting antigens present in the allogeneic cancer vaccines. We observe that distracting antigens greatly outnumber shared TAAs and neoantigens, providing one potential explanation for the lack of observed responses to allogeneic cancer vaccines. This analysis provides additional rationale for the redirection of efforts toward a personalized cancer vaccine approach. Significance: A comprehensive examination of allogeneic cancer vaccine antigen repertoire using large-scale genomics datasets highlights the large number of distracting antigens and argues for more personalized approaches to immunotherapy that leverage recent strategies in tumor antigen identification.

Intentional Modulation of Ibrutinib Pharmacokinetics through CYP3A Inhibition

Ibrutinib (Imbruvica; PCI-32765) is an orally administered inhibitor of Bruton's tyrosine kinase that has transformed the treatment of B-cell malignancies. However, ibrutinib has very low oral bioavailability that contributes to significant variability in systemic exposure between patients, and this has the potential to affect both efficacy and toxicity. We hypothesized that the oral bioavailability of ibrutinib is limited by CYP3A isoform–mediated metabolism, and that this pathway can be inhibited to improve the pharmacokinetic properties of ibrutinib. Pharmacokinetic studies were performed in wild-type mice and mice genetically engineered to lack all CYP3A isoforms (CYP3A−/−) that received ibrutinib alone or in combination with CYP3A inhibitors cobicistat or ketoconazole. Computational modeling was performed to derive doses of ibrutinib that, when given after a CYP3A inhibitor, results in therapeutically relevant drug levels. Deficiency of CYP3A in mice was associated with an approximately 10-fold increase in the AUC of ibrutinib. This result could be phenocopied by administration of cobicistat before ibrutinib in wild-type mice, but cobicistat did not influence levels of ibrutinib in CYP3A−/− mice. Population pharmacokinetic and prospectively validated physiologically based pharmacokinetic models established preclinical and clinical doses of ibrutinib that could be given safely in combination with cobicistat without negatively affecting antileukemic properties. These findings signify a dominant role for CYP3A-mediated metabolism in the elimination of ibrutinib, and suggest a role for pharmacologic inhibitors of this pathway to intentionally modulate the plasma levels and improve the therapeutic use of this clinically important agent. Significance: Ibrutinib has limited oral bioavailability, which contributes to significant interindividual pharmacokinetic variability. Using engineered mouse models, we here report a causal relationship between CYP3A-mediated metabolism and ibrutinib's bioavailability and drug–drug interaction with cobicistat. These results offer a mechanistic basis for reported pharmacokinetic interactions with ibrutinib, and in conjunction with a newly developed computational model, allow for the rational design of clinical trials aimed at improving the therapeutic use of ibrutinib.