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Purpose:To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer.Experimental Design:We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures.Results:We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade–resistant metastatic urothelial cancer.Conclusions:The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139

Supplemental Figure 1. Comparison of the anti-proliferative activity of SMAP across a panel of cell lines. Supplemental Figure 2. The chemical structure of SMAPs utilized in the studies detailed in the manuscript. Supplemental Figure 3. The effects of SMAPs on cell proliferation and cell survival. Supplemental Figure 4. Control studies for co-immunoprecipitation experiments. Supplemental Figure 5. Activity of SMAPs in vivo with the correlation of anti-tumor activity with SMAP-2 exposure in serum of treated mice.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065–80. ©2018 AACR.

Table S1. KSEA Analysis and the Mean FC Method in LNCaP treated with SMAP. Table S2. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated LNCaP colony formation assay. Table S3. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated 22Rv1 colony formation assay. Table S4. ANOVA with multiple comparisons and Dunnett''s post-hoc test for Annexin V staining of LNCap and 22Rv1 cells. Table S5. PPI of the phosphoproteomics dataset. Table S6. Kinase Substrate Database for LNCaP treated with SMAP. Table S7. ANOVA with multiple comparisons and Tukey''s post-hoc test for SMAP treatment in LNCaP cells phosphorylated to total AR ratio protein. Table S8. ANOVA for qRT-PCR RNA for AR targets in LNCaP and 22Rv1 cells in presence of SMAPs. Table S9. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LNCaP and 22Rv1 cells treated with SMAP, bortezomib, and combination. Table S10. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP- Small T stably expressing lines treated with SMAP. Table S11. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP cells in FBS or CSS in presence or absence of R1881. Table S12. ANOVA for qRT-PCR RNA AR targets statistical analysis for LNCaP cells in FBS or CSS in presence or absence of R1881. Table S13. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LnCaP/AR xenograft treatment study: vehicle control, SMAP 400mg/kg, SMAP 100mg/kg and MDV3100 100mg/kg. Table S14. ANOVA with multiple comparisons and Dunnett''s post-hoc test for castrated LNCaP/AR tumor volumes for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg. Table S15. ANOVA with multiple comparisons and Dunnett''s post-hoc test for TUNEL positive staining quantification from castrated LNCaP/AR tumors from individual treatments groups: Vehicle control, SMAP-2 30mg/kg and SMAP-2 100mg/kg. Table S16. ANOVA with multiple comparisons and Dunnett''s post-hoc test for PCNA positive staining quantification from castrated LNCaP/AR for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg.

PDF file - 76K, In MT-RET-1 mouse melanoma cell line we transient trafected with with siRNA-iNOS human, after 48 hours MT-RET-1 mouse cell lysate were subjected to SDS-PAGE and western blot analysis for iNOS, and actin as a loading control.

PDF file - 67K, A375 cells were treated with L-NIL (300 μM and 1000 μM) or the NO scavenger PTIO (300 μM) for 24 hours prior to harvest and preparation of protein lysates. Western Blot analyses of AKT phosphorylation at serine 473 and total AKT demonstrated lack of downregulation of AKT phosphorylation under conditions which strongly reduce downstream mTOR pathway activation.

PDF file - 77K, A375 cells were cultured with the indicated concentrations of the NO donor S-nitroso-acetyl-penicillamine (SNAP), or the NO scavenger PTIO for 16 Hours prior to harvest and preparation of protein lysates.. Biotin switch assay was performed as described in Figure 6, and immunoblotting for BCL-2 and AKT performed after streptavidin pull-down to determine nitrosylated levels of these proteins.

PDF file - 135K, A375 melanoma cells were exposed for 6 hours or 24 Hours to ODQ (3 uM, 10 uM), or the vehicle control dimethylsulfoxide (DMSO). mTOR pathway activation was analyzed by 4EBP1 and p-S6RP western blot. Signals were quantified by densitometry, and the ratio of activated p-4EBP1 and p-S6RP was calculated, normalized against total protein (lower panel). ODQ even at extended time point (24 hours) and high dose (10 uM) had little effect on mTOR pathway activation.

PDF file - 132K, Mel 624 (A) and MeWo cells (B) were transfected with iNOS-specific or scrambled control siRNA and inoculated on the CAM for 5 days prior to photography of macroscopic tumors, histological processing, and trypan exclusion viability assay as described in Figure 1. Each graph represents data derived from 2 independent experiments, and error bars indicate standard error of the mean.

Melanoma is one of the cancers of fastest-rising incidence in the world. Inducible nitric oxide synthase (iNOS) is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K–AKT–mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p70 ribosomal S6 kinase (p-P70S6K), p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of tuberous sclerosis complex (TSC) 2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Ras homolog enriched in brain (Rheb), a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of the mTOR pathway members. Exogenously supplied NO was also sufficient to reverse the mTOR pathway inhibition by the B-Raf inhibitor vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers. Cancer Res; 74(4); 1067–78. ©2014 AACR.

Purpose: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer. Experimental Design: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures. Results: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade–resistant metastatic urothelial cancer. Conclusions: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood. See related commentary by Drake, p. 4139

447 Background: Transurethral resection of bladder tumor (TURBT) plus systemic therapy has been known for decades to achieve durable bladder-intact survival in a subset of patients with MIBC but efforts to advance this paradigm have been complicated by a lack of (a) prospective studies, (b) rigorous approaches to assess and define clinical complete response (cCR), and (c) integration of novel therapies. Methods: Eligible patients were cisplatin-eligible with cT2-T4aN0M0 urothelial bladder cancer. Patients received 4 cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging including urine cytology, MRI/CT of the bladder, cystoscopy and bladder biopsies. Patients achieving a cCR (normal cytology, imaging, and cT0/Ta) were eligible to proceed without cystectomy and receive nivolumab q2 weeks x 8 followed by surveillance. Patients not achieving cCR were recommended to undergo cystectomy. Coprimary endpoints included (1) cCR rate and (2) association between cCR and 2-year outcomes. The key secondary endpoint was the impact of pre-specified baseline genomic alterations on outcomes. Additional biomarkers to refine patient selection were also explored. Results: Between 8/2018-11/2020, 76 patients were enrolled at 7 sites (male 79%, median age 69; cT2 = 56%, cT3 = 32%, cT4 = 12%). Median follow-up is 27 months. 72/76 patients underwent clinical restaging and a cCR was achieved in 33/76 (43%; 95% CI: 32%, 55%). One cCR patient opted for immediate cystectomy (ypTaN0M0). Outcomes are summarized in the Table. Baseline ERCC2, ATM, FANCC, or RB1 alterations were not, but tumor mutational burden ≥ 10 mutations/mb was, significantly associated with the composite endpoint of ypT0 (immediate cystectomy) or 2-year bladder-intact metastasis-free survival (BIMFS). On landmark analysis, VI-RADS (Vesical Imaging–Reporting and Data System) score (3-5 versus 1-2) on restaging MRI (central blinded review) was associated with inferior BIMFS (HR 4.5; p =

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8

Prostate cancer (PCa) preferentially metastasizes to bone, leading to complications including severe pain, fractures, spinal cord compression, bone marrow suppression, and a mortality of ∼70%. In spite of recent advances in chemo-, hormonal, and radiation therapies, bone-metastatic, castrate-resistant PCa is incurable. PCa is somewhat unique among the solid tumors in its tendency to produce osteoblastic lesions composed of hypermineralized bone with multiple layers of poorly organized type I collagen fibrils that have reduced mechanical strength. Many of the signaling pathways that control normal bone homeostasis are at play in pathologic PCa bone metastases, including the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin system. A number of PCa-derived soluble factors have been shown to induce the dysfunctional osteoblastic phenotype. However, therapies directed at these osteoblastic-stimulating proteins have yielded disappointing clinical results to date. One of the soluble factors expressed by PCa cells, particularly in bone metastases, is prostatic acid phosphatase (PAP). Human PAP is a prostate epithelium-specific secretory protein that was the first tumor marker ever described. Biologically, PAP exhibits both phosphatase activity and ecto-5′-nucleotidase activity, generating extracellular phosphate and adenosine as the final products. Accumulating evidence indicates that PAP plays a causal role in the osteoblastic phenotype and aberrant bone mineralization seen in bone-metastatic, castrate-resistant PCa. Targeting PAP may represent a therapeutic approach to improve morbidity and mortality from PCa osteoblastic bone metastases.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is causing the COVID-19 pandemic. There is no current standard of care. Clinicians need to be mindful of the toxicity of a wide variety of possibly unfamiliar substances being tested or repurposed to treat COVID-19. The United States Food and Drug Administration (FDA) has provided emergency authorization for the use of chloroquine and hydroxychloroquine. These two medications may precipitate ventricular dysrhythmias, necessitating cardiac and electrolyte monitoring, and in severe cases, treatment with epinephrine and high-doses of diazepam. Recombinant protein therapeutics may cause serum sickness or immune complex deposition. Nucleic acid vaccines may introduce mutations into the human genome. ACE inhibitors and ibuprofen have been suggested to exacerbate the pathogenesis of COVID-19. Here, we review the use, mechanism of action, and toxicity of proposed COVID-19 therapeutics.

INTRODUCTION: Prostatic acid phosphatase (PAP) is a soluble factor secreted by prostate luminal epithelial cells. PAP expression correlates with prostate cancer (PCa) bone metastases and poor survival. The androgenic regulation of PAP in prostate development and tumorigenesis is not fully understood. We investigated the relationship between PAP and androgens in human prostate specimens and in vivo. HYPOTHESIS AND OBJECTIVES: We hypothesized that PAP expression was independent of androgens. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human PCa bone metastases, and xenograft and surgical castration mouse models. METHODS: Immunohistochemical staining for PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was carried out on human fetal prostate (9.5, 11.5, 13, 16.5, 18 and 20 weeks of gestational age), archival human PCa bone metastases, and PCa mouse models. For xenograft studies, PAP-expressing PCa cell lines, LNCaP, C42B, and VCaP cells, were inoculated subcutaneously into SCID mice. A castration study with surgical or sham castration was performed after VCaP tumors were palpable. Mouse tumor growth and weight were measured biweekly, and tumor tissue isolated after mouse sacrifice. RESULTS: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). However, AR and ERG expression was absent in two of four castrate-resistant specimens. PSA was weakly expressed in human castration-resistant bone metastatic prostate specimens. In vivo, PAP expression was observed in all tumor models; however, the expression of PAP differed among androgen-sensitive models; LNCaP (low PAP), C42B (moderate PAP) and VCaP (high PAP). Castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact mice. Strong expression of PAP was observed after castration. In contrast, AR, PSA, and ERG expression were reduced in castrated VCaP tumors compared to tumors from intact mice. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression located in hypoxic areas near necrosis. CONCLUSIONS: Our findings demonstrated that PAP is expressed early in normal human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenografts and human PCa bone metastases, androgens did not significantly regulate PAP expression. These data demonstrate that PAP is a marker of early progenitor cells in the normal prostate and is persistently expressed after castration. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix–loop–helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non–small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

Introduction: Prostatic Acid Phosphatase (PAP), a protein phosphatase and 5’ecto-nucleotidase, is expressed in prostate cancer (PCa) bone metastases and correlates with poor survival. Growing evidence suggests that PAP is not regulated by androgens, but rather by factors in the tumor microenvironment. Hypothesis: We hypothesized that PAP is a marker for a more progenitor type PCa cell and its expression is androgen-independent, persisting in castration-resistant disease. Methods: Protein expression of PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was assessed with immunohistochemistry in human fetal prostate (9.5 - 20 weeks of gestational age), archival human PCa bone metastases, and human PCa cell lines. VCaP cells were treated in vitro with dihydrotestosterone (DHT) and the effects on AR and PAP protein expression determined with Western Blotting. PAP-expressing PCa cell lines (LNCaP, C42B, and VCaP) were inoculated subcutaneously (s.c.) into SCID mice. To model tumor-bone interaction, LNCaP and MC3T3 osteoblast cells were co-inoculated s.c. into SCID mice. A VCaP castration study with surgical or sham castration was performed after tumors were palpable and effects of castration on tumor growth and protein expression determined. Results: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age prior to PSA and AR expression. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). In vitro, VCaP cells expressed high levels of AR and PAP protein and DHT treatment increased AR and decreased PAP protein expression. In vivo, PAP expression was observed in all tumor models; LNCaP (low PAP expression), C42B (moderate PAP expression) and VCaP (high PAP expression). Castrated VCaP tumors underwent tumor stasis, were significantly smaller compared to intact mice, had decreased AR, PSA and ERG expression but persistent expression of PAP. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression in hypoxic areas near necrosis. Inoculation of LNCaP cells with MC3T3 osteoblastic cells increased PAP expression in vivo. Conclusions: PAP is expressed early in human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenograft tumors and human PCa bone metastases, androgens did not significantly regulate PAP expression. Both hypoxia and stroma increased PAP expression. These data demonstrate that PAP is a marker of early progenitor cells, is persistently expressed after castration and is upregulated by tumor microenvironmental factors. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Fibrous sheath interacting protein 1 (FSIP1), a spermatogenesis-related testicular antigen, is expressed in abundance in breast cancers, particularly in those overexpressing human epidermal growth factor receptor 2 (HER2); however, little is known about its role in regulating the growth and metastasis of breast cancer cells. We and others have shown previously that FSIP1 expression in breast cancer correlates positively with HER2-positivity, recurrence, and metastases and negatively with survival. Here, using coimmunoprecipitation and microscale thermophoresis, we find that FSIP1 binds to the intracellular domain of HER2 directly. We further show that shRNA-induced FSIP1 knockdown in SKBR3 and MCF-7 breast cancer cells inhibits proliferation, stimulates apoptosis, attenuates epithelial–mesenchymal transition, and impairs migration and invasiveness. Consistent with reduced proliferation and enhanced apoptosis, xenotransplantation of SKBR3 cells stably transfected with sh-FSIP1 into nu/nu mice results in reduced tumor volumes compared with sh-NC transplants. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping using sh-FSIP1 gene signature yielded associations with extracellular matrix protein pathways, and a reduction in SNAI2 protein expression was confirmed on Western blot analysis. Complementarily, interrogation of the Connectivity Map using the same gene signature yielded, as top hits, chemicals known to inhibit epithelial–mesenchymal transition, including rapamycin, 17-N-allylamino-17-demethoxygeldanamycin, and LY294002. These compounds phenocopy the effects of sh-FSIP1 on SKBR3 cell viability. Thus, FSIP1 suppression limits oncogenesis and invasiveness in breast cancer cells and, considering its absence in most other tissues, including normal breast, may become a potential target for breast cancer therapy.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

Objective: Adrenal incidentalomas are increasingly detected with the widespread use of thoracic and abdominal imaging. The most common secretory syndrome in adrenal nodules is autonomous cortisol secretion (ACS). Recent data show that even mild cortisol excess is associated with adverse outcomes. The glucocorticoid receptor antagonist mifepristone has been used in patients with overt Cushing syndrome and hyperglycemia. The purpose of our study was to determine the effect of mifepristone on metabolic parameters in patients with ACS and concomitant prediabetes or diabetes. Methods: Eight patients with either unilateral or bilateral adrenal nodules with ACS were included in the study. Fasting laboratory tests including glucose and insulin levels to calculate homeostatic model assessment for insulin resistance (HOMA-IR) were performed at baseline and again after either 3 months (3 patients) or 6 months (5 patients) on mifepristone 300 mg daily treatment. Patients also completed several validated surveys on mood and quality of life at baseline and follow-up. Results: There were significant reductions in fasting glucose measurements and insulin resistance as measured by HOMA-IR in the 6 of 8 study patients in whom these measurements were available (P = .03). Conclusion: This pilot study demonstrates that mifepristone treatment of ACS is associated with a significant decrease in fasting glucose and insulin resistance as measured by HOMA-IR scores. Mifepristone treatment of ACS may be considered as a medical option for patients with ACS due to adrenal adenomas with concomitant abnormal glucose parameters in whom surgical removal is not being considered. Abbreviations: ACS = autonomous cortisol secretion; ACTH = adrenocorticotropic hormone; AI = adrenal incidentaloma; DHEAS = dehydroepiandrosterone sulfate; GR = glucocorticoid receptor; HbA1c = hemoglobin A1c; HOMA-IR = homeostatic model assessment for insulin resistance; ODT = overnight dexamethasone suppression test; QoL = quality of life; STAI = state trait anxiety inventory; TSH = thyroid stimulating hormone; UFC = urinary free cortisol.

INTRODUCTION: Prostatic acid phosphatase (PAP) is an acid phosphatase synthesized in prostate epithelial cells that served as the biochemical diagnostic mainstay for prostate cancer for several decades. There are two forms of PAP, secretory(sPAP) and transmembrane (TM-PAP), that are derived from alternative transcripts of the gene. PAP is highly expressed by epithelial cells of the adult prostate, however, the androgenic regulation of prostatic acid phosphatase is not well understood. HYPOTHESIS AND OBJECTIVES: We hypothesized that androgens do not increase PAP expression. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human bone metastases, and intact and castrated xenograft tumors of human VCaP prostate cancer cells. In addition, the effects of dihydrotestosterone (DHT) onTM- and sPAP expression in vitro were quantified. METHODS: Immunohistochemical staining for PAP, androgen receptor (AR) and Prostate Specific Antigen (PSA) protein expression was carried out on human fetal prostate (9.5, 11.5, 13, 16.5 and 18 weeks of gestational age) and human castration-resistant prostate cancer bone metastases archival specimens obtained with IRB approval. Tissue from human benign prostate and breast cancer metastases were used as controls. For xenograft studies, VCaP cells were inoculated subcutaneously into SCID mice and surgical or sham castration was performed after tumor development. Mouse tumors and weights were measured biweekly and tumor tissue isolated after mouse sacrifice. Isolated tumors were stained for PAP, AR and PSA. RESULTS: PAP expression was an early marker of prostate development and observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was observed in all human castration-resistant prostate cancer bone metastases examined (4/4 specimens). The AR was absent in two of four specimens. PSA was expressed in human castration-resistant bone metastatic prostate specimens; however, the expression was not as strong as PAP. In vivo, castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact animals but increased PAP expression was noted after castration. In contrast, PSA and AR expression was reduced in castrated VCaP tumors compared to tumors from intact mice. DHT treatment of VCaP cells increased AR expression while decreasing both sPAP and TM-PAP at doses ranging from 10-7-10-14M for 24 h. CONCLUSIONS: Our findings demonstrate that PAP is expressed early in normal human fetal prostate development and may be a marker of the early stages of differentiation. DHT decreased the expression of both isoforms of PAP in vitro and castration increased PAP expression in vivo. TM-PAP may serve as a potential target for castration-resistant prostate cancer as its expression increases with castration.

Although tyrosine kinase inhibitors (TKIs) have demonstrated significant efficacy in advanced lung adenocarcinoma (LUAD) patients with pathogenic alterations in EGFR, most patients develop acquired resistance to these agents via mechanisms enabling the sustained activation of the PI3K and MAPK oncogenic pathways downstream of EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the PI3K and MAPK pathways and represents a promising therapeutic strategy for the treatment of TKI-resistant LUAD. After establishing the efficacy of small molecule activators of PP2A (SMAPs) in a transgenic EGFR(L858R) model and TKI-sensitive cell lines, we evaluated their therapeutic potential in vitro and in vivo in TKI-resistant models. PP2A activation resulted in apoptosis, significant tumor growth inhibition, and downregulation of PI3K and MAPK pathways. Combination of SMAPs and TKI afatinib resulted in an enhanced effect on the downregulation of the PI3K pathway via degradation of the PP2A endogenous inhibitor CIP2A. An improved effect on tumor growth inhibition was observed in a TKI-resistant xenograft mouse model treated with a combination of both agents. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.

Background HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. Results To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. Conclusions Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS. Electronic supplementary material The online version of this article (10.1186/s12977-019-0464-3) contains supplementary material, which is available to authorized users.

4503 Background: Transurethral resection of bladder tumor (TURBT) plus systemic therapy has been known for decades to achieve durable bladder-intact survival in a subset of patients with MIBC but efforts to advance this paradigm have been complicated by (a) lack of prospective studies exclusively testing cisplatin-based neoadjuvant chemotherapy, (b) lack of rigorous methods to define clinical complete response (cCR) and its association with long term outcomes and (c) limited understanding of the role of “salvage” cystectomy. Methods: Eligible patients were cisplatin-eligible with cT2-T4aN0M0 urothelial bladder cancer. Patients received 4 cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging including urine cytology, MRI/CT of the bladder, cystoscopy and bladder/prostatic urethral biopsies. Patients achieving a cCR (normal cytology, imaging, and cT0/Ta) were eligible to proceed without cystectomy and receive nivolumab q2 weeks x 8 followed by surveillance; otherwise, patients underwent cystectomy. Coprimary endpoints included (1) cCR rate and (2) ability of cCR to predict 2-year metastasis-free survival (MFS). The key secondary endpoint was the impact of genomic alterations in baseline TURBT (TMB, ERCC2, FANCC, RB1, ATM) on performance of cCR for predicting MFS. The cCR rate coprimary endpoint, and interim analysis of 1-year outcomes, are reported. Results: Between 8/2018-11/2020, 76 patients were enrolled at 7 sites (male 79%, median age 69; cT2 = 56%, cT3 = 32%, cT4 = 12%) and 64 (84%) have completed post-cycle 4 restaging; 31/64 achieved a cCR (48%; 95% CI 36%, 61%). The median follow-up of cCR patients is 13.7 months (range, 2.5-24 months). One cCR patient opted for immediate cystectomy (pTaN0M0). Outcomes for the entire cohort are summarized in the table below. Local recurrence has occurred in 8/31 cCR patients and 6 underwent cystectomy (pT0N0 = 1, pTaN0 = 1, pTisN0 =1, pT2N0 = 2, pT4N1 = 1). TMB ≥ 10 mut/Mb (p=0.02) or mutant ERCC2 (p=0.02) were associated with cCR or pT0. Conclusions: TURBT + gemcitabine, cisplatin, plus nivolumab achieves stringently defined cCR in a large subset of patients with MIBC. 1-year bladder intact survival is possible though the durability of responses, and role of genomic biomarkers in management algorithms, requires longer follow-up. Clinical trial information: NCT03558087. [Table: see text]

Prostate cancer (PCa) is unique in its tendency to produce osteoblastic (OB) bone metastases. There are no existing therapies that specifically target the OB phase that affects 90% of men with bone metastatic disease. Prostatic acid phosphatase (PAP) is secreted by PCa cells in OB metastases and increases OB growth, differentiation, and bone mineralization. The purpose of this study was to investigate whether PAP effects on OB bone metastases are mediated by autocrine and/or paracrine alterations in the receptor activator of nuclear factor κ-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system. To investigate whether PAP modulated these factors and altered the bone reaction, we knocked down PAP expression in VCaP cells and stably overexpressed PAP in PC3M cells, both derived from human PCa bone metastases. We show that knockdown of PAP in VCaP cells decreased OPG while increasing RANK/RANKL expression. Forced overexpression of PAP in PC3M cells had the inverse effect, increasing OPG while decreasing RANK/RANKL expression. Coculture of PCa cells with MC3T3 preosteoblasts also revealed a role for secretory PAP in OB-PCa cross talk. Reduced PAP expression in VCaP cells decreased MC3T3 proliferation and differentiation and reduced their OPG expression. PAP overexpression in PC3M cells altered the bone phenotype creating OB rather than osteolytic lesions in vivo using an intratibial model. These findings demonstrate that PAP secreted by PCa cells in OB bone metastases increases OPG and plays a critical role in the vicious cross talk between cancer and bone cells. These data suggest that inhibition of secretory PAP may be an effective strategy for PCa OB bone lesions.

The natural history and optimal management strategy for men with human immunodeficiency virus (HIV) and prostate cancer remain to be definitively characterized. This study was conducted to evaluate the clinical characteristics and outcomes of HIV-seropositive men treated with robotic-assisted radical laparoscopic prostatectomy for localized prostate cancer.After Institutional Review Board approval, a prospective database of 2175 operative cases of clinically localized prostate adenocarcinoma was reviewed. Thirteen patients were identified as HIV-positive. Tumor characteristics, operative outcomes, postoperative outcomes, histology (Gleason score), local invasion, biochemical recurrence, and surgical complications were compared with HIV-negative patients.There were no preoperative demographic differences between the HIV-positive and HIV-negative patients. HIV-positive patients had higher prostate specific antigen (PSA) levels at time of diagnosis which was not statistically significant. However, HIV-positive patients had higher D'Amico risk assessment (p 0.05). There was no postoperative complication. HIV-positive patients treated with robotic prostatectomy had similarly favorable perioperative and short-term biochemical recurrence-free survival outcomes.Our findings show that minimally invasive prostatectomy can be safely considered as a therapeutic option in otherwise eligible HIV-positive patients with clinically significant prostate cancer. Further research is necessary to outline a diagnostic and treatment guideline for HIV-positive men in detection and treatment of prostate cancer.

Development of molecularly targeted therapies have shifted our clinical approach to cancer allowing for an integrated treatment approach where tumor genotype and molecular profiles are paired in a clinically actionable manner with specific targeted therapies. This approach maximizes efficacy and minimizes toxicity for each patient. While the vast majority of molecularly targeted therapies have focused on the development of kinase inhibition, an equally effective yet underexplored strategy exists in the activation or regulation of phosphatases. The tumor suppressor Protein Phosphatase 2A (PP2A) negatively regulates multiple oncogenic drivers common to a broad range of human malignancies including MYC, AKT, and ERK; and has been demonstrated as a critical tumor suppressor. In cancer, PP2A is inactivated through several mechanisms including increased expression of endogenous inhibitors, post-translational modifications of the catalytic subunit, and somatic mutation in its catalytic, regulatory or scaffold subunits. The vast mechanisms employed to dysregulate and inhibit PP2A in cancer, highlights it's potential as an endogenous molecular target to combat malignant growth. Our laboratory has been involved in the development of a novel series of small molecule activators of PP2A (SMAPs). Using structural, biochemical, cellular, and in vivo model systems we reveal how the lead SMAP, DT-061, specifically stabilizes a B56 containing PP2A holoenzyme, in its fully assembled, active state, to direct enzymatic activity towards specific oncogenic targets, including its well-known substrate c-MYC. Our structure identifies a unique interfacial small molecule binding pocket that involves all three PP2A subunits to prevent dissociation of select B56 subunit containing holoenzymes. Elucidating the mechanism of SMAPs allowed us to explore appropriate clinical placement for this targeting strategy and investigate potential mechanisms of resistance. For example, in vivo xenograft studies of cancer-associated Aα mutants, most notably R183W, demonstrates that these mutations render malignant cells resistant to these SMAPs and further support the mechanism of SMAPs described. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets. Citation Format: Daniel Leonard, Wei Huang, Sudeh Izadmehr, Caitlin M. O'Connor, Danica D. Wiredja, Zhizhi Wang, Nilesh Zaware, Daniela M. Schlatzer, Stefan Schuchner, Janna Kiselar, Yinghua Chen, Nikhil Vasireddi, Abbey L. Perl, Matthew D. Glasky, Wenqing Xu, David L. Brautigan, Egon Ogris, Derek J. Taylor, Goutham Narla. Biased activation of PP2A through selective heterotrimer binding and stabilization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2981.

Summary Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory “B” subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 A structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Kinase inhibitor resistance constitutes a major unresolved clinical challenge in cancer. Furthermore, the role of serine/threonine phosphatase deregulation as a potential cause for resistance to kinase inhibitors has not been thoroughly addressed. We characterize protein phosphatase 2A (PP2A) activity as a global determinant of KRAS-mutant lung cancer cell resistance across a library of >200 kinase inhibitors. The results show that PP2A activity modulation alters cancer cell sensitivities to a large number of kinase inhibitors. Specifically, PP2A inhibition ablated mitogen-activated protein kinase kinase (MEK) inhibitor response through the collateral activation of AKT/mammalian target of rapamycin (mTOR) signaling. Combination of mTOR and MEK inhibitors induced cytotoxicity in PP2A-inhibited cells, but even this drug combination could not abrogate MYC up-regulation in PP2A-inhibited cells. Treatment with an orally bioavailable small-molecule activator of PP2A DT-061, in combination with the MEK inhibitor AZD6244, resulted in suppression of both p-AKT and MYC, as well as tumor regression in two KRAS-driven lung cancer mouse models. DT-061 therapy also abrogated MYC-driven tumorigenesis. These data demonstrate that PP2A deregulation drives MEK inhibitor resistance in KRAS-mutant cells. These results emphasize the need for better understanding of phosphatases as key modulators of cancer therapy responses.

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment. Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065–80. ©2018 AACR.

In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.