Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis.

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

18 F-sodium fluoride positron emission tomography quantitation of bone metastases in African American and non-African American men with metastatic prostate cancer.

BACKGROUND: Bone is the most common site of metastases in men with prostate cancer. The objective of this study was to explore potential racial differences in the distribution of tumor metastases in the axial and appendicular skeleton. METHODS: We conducted a retrospective review of patients with metastatic prostate cancer to the bone as detected by 18 F-sodium fluoride positron emission tomography/computed tomography (18 F-NaF PET/CT) scans. In addition to describing patients' demographics and clinical characteristics, the metastatic bone lesions, and healthy bone regions were detected and quantified volumetrically using a quantitative imaging platform (TRAQinform IQ, AIQ Solutions). RESULTS: Forty men met the inclusion criteria with 17 (42%) identifying as African Americans and 23 (58%) identifying as non-African Americans. Most of the patients had axial (skull, ribcage, and spine) disease. The location and the number of lesions in the skeleton of metastatic prostate cancer patients with low disease burden were not different by race. CONCLUSIONS: In low-disease burden patients with metastatic prostate cancer, there were no overall differences by race in the location and number of lesions in axial or appendicular skeleton. Therefore, given equal access to molecular imaging, African Americans might derive similar benefits. Whether this holds true for patients with a higher disease burden or for other molecular imaging techniques is a topic for further study.

Alcohol dehydrogenase expression patterns in normal prostate, benign prostatic hyperplasia, and prostatic adenocarcinoma in African American and Caucasian men.

BACKGROUND: In situ metabolism of ethanol by alcohol dehydrogenases (ADHs) contributes to oxidative damage of cells and DNA and has been linked to carcinogenesis in numerous epithelial tissues. The goal of this study was to determine expression patterns of ADH1 and ADH7 isozymes in normal, hyperplastic (benign prostatic hyperplasia [BPH]) and neoplastic (prostate cancer [PCa]) prostate. Furthermore, racial differences in ADH expression between African Americans and Caucasians were investigated. METHODS: ADH expression patterns were characterized by density analysis of ADH immunohistochemistry (n = 21) and real-time RT-PCR of total RNAs by laser-capture microdissection (n = 10) and whole tissue formalin-fixed paraffin embedded prostate biopsies (n = 63). RESULTS: ADH protein is found in normal prostate and is primarily associated with glandular epithelium. Transcripts of ADH1B are suppressed in PCa compared to BPH (p = 0.0095). Racial differences in ADH7 transcripts exist between African American and Caucasian men. A total of 57.6% of biopsies from African American prostates have detectable ADH7 messenger RNA (mRNA) transcripts compared to the 13.3% of Caucasian prostate biopsies with detectable transcripts (p = 0.0005). This increased frequency of detection contributes to higher mean ADH7 mRNA transcript levels in African Americans (p = 0.001). CONCLUSIONS: To our knowledge this study is the first to report downregulation of ADH1B in neoplastic prostate at the transcriptional level, suggesting protective regulatory functions. ADH7 transcripts were not detectable in all samples and was found in higher frequency and amount in our African American samples. Racial differences in ADH7 within the prostate is a novel finding and should be investigated further.

Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma.

Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.

ONC201 induces the unfolded protein response (UPR) in high- and low-grade ovarian carcinoma cell lines and leads to cell death regardless of platinum sensitivity.

OBJECTIVES: Treatment of both platinum resistant high grade (HG) and low-grade (LG) ovarian cancer (OVCA) poses significant challenges as neither respond well to conventional chemotherapy leading to morbidity and mortality. Identification of novel agents that can overcome chemoresistance is therefore critical. Previously, we have demonstrated that OVCA has basal upregulated unfolded protein response (UPR) and that targeting cellular processes leading to further and persistent upregulation of UPR leads to cell death. ONC201 is an orally bioavailable Dopamine Receptor D2 inhibitor demonstrating anticancer activity and was found to induce UPR. Given its unique properties, we hypothesized that ONC201 would overcome platinum resistance in OVCA. METHODS: Cisplatin sensitive and resistant HG OVCA and two primary LG OVCA cell lines were studied. Cell viability was determined using MTT assay. Cell migration was studied using wound healing assay. Apoptosis and mitochondrial membrane potential were investigated using flow cytometry. Analysis of pathway inhibition was performed by Western Blot. mRNA expression of UPR related genes were measured by qPCR. In vivo studies were completed utilizing axillary xenograft models. Co-testing with conventional chemotherapy was performed to study synergy. RESULTS: ONC201 significantly inhibited cell viability and migration in a dose dependent manner with IC50's from 1-20 µM for both cisplatin sensitive and resistant HG and LG-OVCA cell lines. ONC201 lead to upregulation of the pro-apoptotic arm of the UPR, specifically ATF-4/CHOP/ATF3 and increased the intrinsic apoptosispathway. The compensatory, pro-survival PI3K/AKT/mTOR pathway was downregulated. In vivo, weekly dosing of single agent ONC201 decreased xenograft tumor size by ~50% compared to vehicle. ONC201 also demonstrated significant synergy with paclitaxel in a highly platinum resistant OVCA cell-line (OV433). CONCLUSIONS: Our findings demonstrate that ONC201 can effectively overcome chemoresistance in OVCA cells by blocking pro-survival pathways and inducing the apoptotic arm of the UPR. This is a promising, orallybioavailable therapeutic agent to consider in clinical trials for patients with both HG and LG OVCA.

Inflammation markers on benign breast biopsy are associated with risk of invasive breast cancer in African American women.

PURPOSE: Markers of inflammation, including crown-like structures of the breast (CLS-B) and infiltrating lymphocytes (IL), have been identified in breast tissue and associated with increased risk of breast cancer (BrCa), however most of this work has been performed in primarily non-Hispanic white women. Here, we examined whether CLS-B and IL are associated with invasive BrCa in African American (AA) women. METHODS: We assessed breast biopsies from three 5-year age-matched groups: BrCa-free AA women (50 Volunteer) from the Komen Normal Tissue Bank (KTB) and AA women with a clinically-indicated biopsy diagnosed with benign breast disease (BBD) from our Detroit cohort who developed BrCa (55 BBD-cancer) or did not develop BrCa (47 BBD only, year of biopsy matched to BBD-cancer). Mean adipocyte diameter and total adipose area were estimated from digital images using the Adiposoft plugin from ImageJ. Associations between CLS-B, IL, and BrCa among KTB and Detroit biopsies were assessed using multivariable multinomial and conditional logistic regression models. RESULTS: Among all biopsies, Volunteer and BBD only biopsies did not harbor CLS-B or IL at significantly different rates after adjusting for logarithm of adipocyte area, adipocyte diameter, and BMI. Among clinically-indicated BBD biopsies, BBD-cancer biopsies were more likely to exhibit CLS-B (odds ratio (OR) = 3.36, 95% Confidence Interval (CI): 1.33-8.48) or IL (OR = 4.95, 95% CI 1.76-13.9) than BBD only biopsies after adjusting for total adipocyte area, adipocyte diameter, proliferative disease, and BMI. CONCLUSIONS: CLS-B and IL may serve as histological markers of BrCa risk in benign breast biopsies from AA women.

Role of TET1 and 5hmC in an Obesity-Linked Pathway Driving Cancer Stem Cells in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptor, progesterone receptor, and the HER2 but is enriched with cancer stem cell-like cells (CSC). CSCs are the fraction of cancer cells recognized as the source of primary malignant tumors that also give rise to metastatic recurrence. 5-Hydroxymethylcytosine (5hmC) is a DNA epigenetic feature derived from 5-methylcytosine by action of tet methylcytosine dioxygenase enzymes (e.g., TET1); and although TET1 and 5hmC are required to maintain embryonic stem cells, the mechanism and role in CSCs remain unknown. Data presented in this report support the conclusion that TET1 and TET1-dependent 5hmC mediate hydrogen peroxide (H2O2)-dependent activation of a novel gene expression cascade driving self-renewal and expansion of CSCs in TNBC. Evidence presented also supports that the H2O2 affecting this pathway arises due to endogenous mechanisms-including downregulation of antioxidant enzyme catalase in TNBC cells-and by exogenous routes, such as systemic inflammation and oxidative stress coupled with obesity, a known risk factor for TNBC incidence and recurrence. IMPLICATIONS: This study elucidates a pathway dependent on H2O2 and linked to obesity-driven TNBC tumor-initiating CSCs; thus, it provides new understanding that may advance TNBC prevention and treatment strategies.

Heritable Susceptibility to Breast Cancer among African-American Women in the Detroit Research on Cancer Survivors Study.

BACKGROUND: African-American women have high rates of breast cancer associated with hereditary features. However, no studies have reported the prevalence of inherited variation across all genes known to be breast cancer risk factors among African-American patients with breast cancer not selected for high-risk characteristics. METHODS: We evaluated 182 African-American women diagnosed with invasive breast cancer in metropolitan Detroit via targeted capture and multiplex sequencing of 13 well-established breast cancer risk genes and five suggested breast cancer risk genes. RESULTS: We identified 24 pathogenic variants in 23 women [12.6%; 95% confidence interval (CI), 8.2%-18.4%] and five genes (BRCA2, BRCA1, ATM, RAD50, CDH1). BRCA1 and BRCA2 accounted for 58.3% of all pathogenic variants. An additional six pathogenic variants were found in suggested breast cancer risk genes (MSH6, MUTYH, NF1, BRIP1). CONCLUSIONS: The prevalence of germline pathogenic variants is relatively high among African-American patients with breast cancer unselected for high-risk characteristics across a broad spectrum of genes. IMPACT: This study helps to define the genomic landscape of breast cancer susceptibility in African-American women who could benefit from enhanced surveillance and screening.

CLCA2 expression is associated with survival among African American women with triple negative breast cancer.

PURPOSE: Black/African American (AA) women are twice as likely to be diagnosed with triple negative breast cancer (TNBC) compared to whites, an aggressive breast cancer subtype associated with poor prognosis. There are no routinely used targeted clinical therapies for TNBC; thus there is a clear need to identify prognostic markers and potential therapeutic targets. METHODS: We evaluated expression of 27,016 genes in 155 treatment-naïve TN tumors from AA women in Detroit. Associations with survival were evaluated using Cox proportional hazards models adjusting for stage and age at diagnosis, and p-values were corrected using a false discovery rate. Our validation sample consisted of 494 TN tumors using four publically available data sets. Meta-analyses were performed using summary statistics from the four validation results. RESULTS: In the Detroit AA cohort, CLCA2 [Hazard ratio (HR) = 1.56, 95% confidence interval (CI) 1.31-1.86, nominal p = 5.1x10-7, FDR p = 0.014], SPIC [HR = 1.47, 95%CI 1.26-1.73, nominal p = 1.8x10-6, FDR p = 0.022], and MIR4311 [HR = 1.57, 95% CI 1.31-1.92, nominal p = 2.5x10-5, FDR p = 0.022] expression were associated with overall survival. Further adjustment for treatment and breast cancer specific survival analysis did not substantially alter effect estimates. CLCA2 was also associated with increased risk of death in the validation cohorts [HR = 1.14, 95% CI 1.05-1.24, p = 0.038, p-heterogeneity = 0.88]. CONCLUSIONS: We identified CLCA2 as a potential prognostic marker for TNBC in AA women.

Cetuximab PET delineated changes in cellular distribution of EGFR upon dasatinib treatment in triple negative breast cancer.

BACKGROUND: At least 50% of triple negative breast cancer (TNBC) overexpress the epidermal growth factor receptor, EGFR, which paved the way for clinical trials investigating its blockade. Outcomes remained dismal stemming from mechanisms of resistance particularly the nuclear cycling of EGFR, which is enhanced by Src activation. Attenuation of Src reversed nuclear translocation, restoring EGFR to the cell surface. Herein, we hypothesize that changes in cellular distribution of EGFR upon Src inhibition with dasatinib can be annotated through the EGFR immunopositron emission tomography (immunoPET) radiotracer, [89Zr]Zr-cetuximab. METHODS: Nuclear and non-nuclear EGFR levels of dasatinib-treated vs. untreated MDA-MB-231 and MDA-MB-468 cells were analyzed via immunoblots. Both treated and untreated cells were exposed to [89Zr]Zr-cetuximab to assess binding at 4 °C and 37 °C. EGFR-positive MDA-MB-231, MDA-MB-468, and a patient-derived xenograft were treated with dasatinib or vehicle followed by cetuximab PET imaging to compare EGFR levels. After imaging, the treated mice were separated into two groups: one cohort continued with dasatinib with the addition of cetuximab while the other cohort received dasatinib alone. Correlations between the radiotracer uptake vs. changes in tumor growth and EGFR expression from immunoblots were analyzed. RESULTS: Treated cells displayed higher binding of [89Zr]Zr-cetuximab to the cell membrane at 4 °C and with greater internalized activity at 37 °C vs. untreated cells. In all tumor models, higher accumulation of the radiotracer in dasatinib-treated groups was observed compared to untreated tumors. Treated tumors displayed significantly decreased pSrc (Y416) with retained total Src levels compared to control. In MDA-MB-468 and PDX tumors, the analysis of cetuximab PET vs. changes in tumor volume showed an inverse relationship where high tracer uptake in the tumor demonstrated minimal tumor volume progression. Furthermore, combined cetuximab and dasatinib treatment showed better tumor regression compared to control and dasatinib-only-treated groups. No benefit was achieved in MDA-MB-231 xenografts with the addition of cetuximab, likely due to its KRAS-mutated status. CONCLUSIONS: Cetuximab PET can monitor effects of dasatinib on EGFR cellular distribution and potentially inform treatment response in wild-type KRAS TNBC.

Biomarkers and Bone Imaging Dynamics Associated with Clinical Outcomes of Oral Cabozantinib Therapy in Metastatic Castrate-Resistant Prostate Cancer.

PURPOSE: Cabozantinib is a multitargeted tyrosine kinase inhibitor that demonstrated remarkable responses on bone scan in metastatic prostate cancer. Randomized trials failed to demonstrate statistically significant overall survival (OS). We studied the dynamics of biomarker changes with imaging and biopsies pretherapy and posttherapy to explore factors that are likely to be predictive of efficacy with cabozantinib.Experimental Design: Eligibility included patients with metastatic castrate-resistant prostate cancer with normal organ function and performance status 0-2. Cabozantinib 60 mg orally was administered daily. Pretherapy and 2 weeks post, 99mTc-labeled bone scans, positron emission tomography with 18F-sodium fluoride (NaF-PET) and 18F-(1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl) thymine (FMAU PET) scans were conducted. Pretherapy and posttherapy tumor biopsies were conducted, and serum and urine bone markers were measured. RESULTS: Twenty evaluable patients were treated. Eight patients had a PSA decline, of which 2 had a decline of ≥50%. Median progression-free survival (PFS) and OS were 4.1 and 11.2 months, respectively, and 3 patients were on therapy for 8, 10, and 13 months. The NaF-PET demonstrated a median decline in SUVmax of -56% (range, -85 to -5%, n = 11) and -41% (range, -60 to -25%, n = 9) for patients who were clinically stable and remained on therapy for ≥4 or <4 cycles, respectively. The FMAU PET demonstrated a median decline in SUVmax of -44% (-60 to -14%) and -42% (-63% to -23%) for these groups. The changes in bone markers and mesenchymal epithelial transition/MET testing did not correlate with clinical benefit. CONCLUSIONS: Early changes in imaging and tissue or serum/urine biomarkers did not demonstrate utility in predicting clinical benefit with cabozantinib therapy.

Downregulation of Rap1Gap: A Switch from DCIS to Invasive Breast Carcinoma via ERK/MAPK Activation.

Diagnosis of breast ductal carcinoma in situ (DCIS) presents a challenge since we cannot yet distinguish those cases that would remain indolent and not require aggressive treatment from cases that may progress to invasive ductal cancer (IDC). The purpose of this study is to determine the role of Rap1Gap, a GTPase activating protein, in the progression from DCIS to IDC. Immunohistochemistry (IHC) analysis of samples from breast cancer patients shows an increase in Rap1Gap expression in DCIS compared to normal breast tissue and IDCs. In order to study the mechanisms of malignant progression, we employed an in vitro three-dimensional (3D) model that more accurately recapitulates both structural and functional cues of breast tissue. Immunoblotting results show that Rap1Gap levels in MCF10.Ca1D cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS (a model of DCIS). Retroviral silencing of Rap1Gap in MCF10.DCIS cells activated extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK), induced extensive cytoskeletal reorganization and acquisition of mesenchymal phenotype, and enhanced invasion. Enforced reexpression of Rap1Gap in MCF10.DCIS-Rap1GapshRNA cells reduced Rap1 activity and reversed the mesenchymal phenotype. Similarly, introduction of dominant negative Rap1A mutant (Rap1A-N17) in DCIS-Rap1Gap shRNA cells caused a reversion to nonmalignant phenotype. Conversely, expression of constitutively active Rap1A mutant (Rap1A-V12) in noninvasive MCF10.DCIS cells led to phenotypic changes that were reminiscent of Rap1Gap knockdown. Thus, reduction of Rap1Gap in DCIS is a potential switch for progression to an invasive phenotype. The Graphical Abstract summarizes these findings.

Phase 0 Trial of AZD1775 in First-Recurrence Glioblastoma Patients.

Purpose: AZD1775 is a first-in-class Wee1 inhibitor with dual function as a DNA damage sensitizer and cytotoxic agent. A phase I study of AZD1775 for solid tumors suggested activity against brain tumors, but a preclinical study indicated minimal blood-brain barrier penetration in mice. To resolve this controversy, we examined the pharmacokinetics and pharmacodynamics of AZD1775 in patients with first-recurrence, glioblastoma.Patients and Methods: Twenty adult patients received a single dose of AZD1775 prior to tumor resection and enrolled in either a dose-escalation arm or a time-escalation arm. Sparse pharmacokinetic blood samples were collected, and contrast-enhancing tumor samples were collected intraoperatively. AZD1775 total and unbound concentrations were determined by a validated LC/MS-MS method. Population pharmacokinetic analysis was performed to characterize AZD1775 plasma pharmacokinetic profiles. Pharmacodynamic endpoints were compared to matched archival tissue.Results: The AZD1775 plasma concentration-time profile following a single oral dose in patients with glioblastoma was well-described by a one-compartment model. Glomerular filtration rate was identified as a significant covariate on AZD1775 apparent clearance. AZD1775 showed good brain tumor penetration, with a median unbound tumor-to-plasma concentration ratio of 3.2, and achieved potential pharmacologically active tumor concentrations. Wee1 pathway suppression was inferred by abrogation of G2 arrest, intensified double-strand DNA breakage, and programmed cell death. No drug-related adverse events were associated with this study.Conclusions: In contrast to recent preclinical data, our phase 0 study of AZD 1775 in recurrent glioblastoma indicates good human brain tumor penetration, provides the first evidence of clinical biological activity in human glioblastoma, and confirms the utility of phase 0 trials as part of an accelerated paradigm for drug development in patients with glioma. Clin Cancer Res; 24(16); 3820-8. ©2018 AACRSee related commentary by Vogelbaum, p. 3790.

Phase II Study of Maintenance Pembrolizumab in Patients with Extensive-Stage Small Cell Lung Cancer (SCLC).

OBJECTIVE: The aim of this study was to assess the efficacy of maintenance pembrolizumab in patients with extensive-stage SCLC after treatment with platinum and etoposide. METHODS: Patients with extensive-stage SCLC with a response or stable disease after induction chemotherapy were eligible. Pembrolizumab at a dose of 200 mg administered intravenously every 3 weeks was initiated within 8 weeks of the last cycle of chemotherapy. The primary end point of the study was progression-free survival (PFS) from study registration, with overall survival (OS) as a key secondary end point. Available tumor tissue was assessed for expression of programmed death ligand 1 (PD-L1) both in the tumor cells and in the surrounding stroma. Blood for circulating tumor cells was collected before the first, second, and third cycles of pembrolizumab. RESULTS: Of the 45 patients enrolled, 56% were male and 22% had treated brain metastases. The median PFS was 1.4 months (95% confidence interval [CI]: 1.3-2.8), with a 1-year PFS of 13%. The median OS was 9.6 months (95% CI: 7.0-12), with a 1-year OS of 37%. Of the 30 tumors that could be assessed, three had PD-L1 expression (≥1%) in the tumor cells. A total of 20 tumors could be assessed for PD-L1 expression in the stroma. The median PFS in the eight patients with tumors positive for expression of PD-L1 at the stromal interface was 6.5 months (95% CI: 1.1-12.8) compared with 1.3 months (95% CI: 0.6-2.5) in 12 patients with tumors negative for this marker. No unexpected toxicities were observed. CONCLUSION: Maintenance pembrolizumab did not appear to improve median PFS compared with the historical data. However, the 1-year PFS rate of 13% and OS rate of 37% suggest that a subset of patients did benefit from pembrolizumab.

Heterogeneity of Human Breast Stem and Progenitor Cells as Revealed by Transcriptional Profiling.

During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/- or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH-CD44+CD24- human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24- by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity.

Phase I Trial of the Combination of Docetaxel, Prednisone, and Pasireotide in Metastatic Castrate-Resistant Prostate Cancer.

BACKGROUND: Pasireotide (SOM230; Novartis Inc, Basel, Switzerland) is a multitargeted somatostatin receptor analogue likely to treat the neuroendocrine, and docetaxel resistant components within metastatic castrate-resistant prostate cancer (mCRPC). This phase I trial tested the combination of pasireotide, docetaxel, and prednisone in pretreated mCRPC. PATIENTS AND METHODS: Chemotherapy naive mCRPC patients received docetaxel 75 mg/m2 intravenously every 21 days and pasireotide intramuscularly every 28 days at escalating dose levels of 40, 60, and 80 mg. Maximum tolerated dose and recommended phase II dose (RP2D) were assessed. RESULTS: Eighteen patients were enrolled with a median age of 65 (range, 49-75) years, and pretherapy prostate-specific antigen of 259.9 ng/mL. The dose-limiting toxicities were Grade 4 hyperglycemia unresponsive to therapy and Grade 4 neutropenia lasting for > 7 days in 1 patient each occurring at the 80-mg dose level of pasireotide. The RP2D was determined at 60 mg every 28 days. Four patients at the 60 mg dose had Grade 3 or 4 hyperglycemia, which responded adequately to therapy. Median time to progression and survival were 7.2 and 18.3 months, respectively. Three of 6 patients with circulating tumor cells ≥5 converted to circulating tumor cells < 5 post therapy. The insulin like growth factor-1 levels revealed a median 51% decrease after therapy. The neuron-specific enolase and chromogranin did not show any marked change. CONCLUSION: The addition of pasireotide to docetaxel and prednisone is clinically feasible at a dose level of 60 mg every 28 days. The combination showed potential for clinical efficacy but needs to be compared with the standard docetaxel and prednisone regimen.

Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity.

Among breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prog-nosis. TNBC does not express estrogen receptor-alpha, progesterone receptor, or the HER2 oncogene; therefore, TNBC lacks targets for molecularly-guided therapies. The concept that EGFR oncogene inhibitor drugs could be used as targeted treatment against TNBC has been put forth based on estimates that 30-60% of TNBC express high levels of EGFR. However, results from clinical trials testing EGFR inhibitors, alone or in combination with cytotoxic chemotherapy, did not improve patient outcomes. Results herein offer an explanation as to why EGFR inhibitors failed TNBC patients and support how combining a select antioxidant and an EGFR-specific small molecule kinase inhibitor (SMKI) could be an effective, novel therapeutic strategy. Treatment with CAT-SKL-a re-engineered protein form of the antioxidant enzyme catalase-inhibited cancer stem-like cells (CSCs), and treatment with the EGFR-specific SMKI erlotinib inhibited non-CSCs. Thus, combining the antioxidant CAT-SKL with erlotinib targeted both CSCs and bulk cancer cells in cultures of EGFR-expressing TNBC-derived cells. We also report evidence that the mechanism for CAT-SKL inhibition of CSCs may depend on antioxidant-induced downregulation of a short alternative mRNA splicing variant of the methyl-CpG binding domain 2 gene, isoform MBD2c.

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers.

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients.

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs.