Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate-resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage plasticity and phenotypic heterogeneity. Castrate-resistant prostate adenocarcinoma can transition to neuroendocrine (NE) and occasionally to amphicrine, co-expressed luminal and NE, phenotypes. We developed castrate-resistant prostate cancer (CRPC) patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and NE phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor (St/Pr) cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent St/Pr cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from St/Pr cells and self-renewing differentiated luminal cells. Neuroendocrine prostate cancer (NEPC) was composed almost exclusively of self-renewing St/Pr cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine St/Pr subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell (SC) characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate SCs.

Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells.

The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype.

Coordinated repression of cell cycle genes by KDM5A and E2F4 during differentiation.

Epigenetic regulation underlies the robust changes in gene expression that occur during development. How precisely epigenetic enzymes contribute to development and differentiation processes is largely unclear. Here we show that one of the enzymes that removes the activating epigenetic mark of trimethylated lysine 4 on histone H3, lysine (K)-specific demethylase 5A (KDM5A), reinforces the effects of the retinoblastoma (RB) family of transcriptional repressors on differentiation. Global location analysis showed that KDM5A cooccupies a substantial portion of target genes with the E2F4 transcription factor. During ES cell differentiation, knockout of KDM5A resulted in derepression of multiple genomic loci that are targets of KDM5A, denoting a direct regulatory function. In terminally differentiated cells, common KDM5A and E2F4 gene targets were bound by the pRB-related protein p130, a DREAM complex component. KDM5A was recruited to the transcription start site regions independently of E2F4; however, it cooperated with E2F4 to promote a state of deepened repression at cell cycle genes during differentiation. These findings reveal a critical role of H3K4 demethylation by KDM5A in the transcriptional silencing of genes that are suppressed by RB family members in differentiated cells.

Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.

Aberrations in epigenetic processes, such as histone methylation, can cause cancer. Retinoblastoma binding protein 2 (RBP2; also called JARID1A or KDM5A) can demethylate tri- and dimethylated lysine 4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by the retinoblastoma protein (pRB). Here, we show that genetic ablation of Rbp2 decreases tumor formation and prolongs survival in Rb1(+/-) mice and Men1-defective mice. These studies link RBP2 histone demethylase activity to tumorigenesis and nominate RBP2 as a potential target for cancer therapy.

Targeting the PI3K/AKT Pathway Overcomes Enzalutamide Resistance by Inhibiting Induction of the Glucocorticoid Receptor.

The PI3K-AKT pathway has pleiotropic effects and its inhibition has long been of interest in the management of prostate cancer, where a compensatory increase in PI3K signaling has been reported following androgen receptor (AR) blockade. Prostate cancer cells can also bypass AR blockade through induction of other hormone receptors, in particular the glucocorticoid receptor (GR). Here we demonstrate that AKT inhibition significantly decreases cell proliferation through both cytostatic and cytotoxic effects. The cytotoxic effect is enhanced by AR inhibition and is most pronounced in models that induce compensatory GR expression. AKT inhibition increases canonical AR activity and remodels the chromatin landscape, decreasing enhancer interaction at the GR gene (NR3C1) locus. Importantly, it blocks induction of GR expression and activity following AR blockade. This is confirmed in multiple in vivo models, where AKT inhibition of established xenografts leads to increased canonical AR activity, decreased GR expression, and marked antitumor activity. Overall, our results demonstrate that inhibition of the PI3K/AKT pathway can block GR activity and overcome GR-mediated resistance to AR-targeted therapy. Ipatasertib is currently in clinical development, and GR induction may be a biomarker to identify responsive patients or a responsive disease state.

The Indenoisoquinoline TOP1 Inhibitors Selectively Target Homologous Recombination-Deficient and Schlafen 11-Positive Cancer Cells and Synergize with Olaparib.

PURPOSE: Irinotecan and topotecan are used to treat a variety of different cancers. However, they have limitations, including chemical instability and severe side effects. To overcome these limitations, we developed the clinical indenoisoquinolines: LMP400 (indotecan), LMP776 (indimitecan), and LMP744. The purpose of the study is to build the molecular rationale for phase II clinical trials. EXPERIMENTAL DESIGN: CellMinerCDB (http://discover.nci.nih.gov/cellminercdb) was used to mine the cancer cell lines genomic databases. The causality of Schlafen11 (SLFN11) was validated in isogenic cell lines. Because topoisomerase I (TOP1)-mediated replication DNA damage is repaired by homologous recombination (HR), we tested the "synthetic lethality" of HR-deficient (HRD) cells. Survival and cell-cycle alterations were performed after drug treatments in isogenic DT40, DLD1, and OVCAR cell lines with BRCA1, BRCA2, or PALB2 deficiencies and in organoids cultured from prostate cancer patient-derived xenografts with BRCA2 loss. We also used an ovarian orthotopic allograft model with BRCA1 loss to validate the efficacy of LMP400 and olaparib combination. RESULTS: CellMinerCDB reveals that SLFN11, which kills cells undergoing replicative stress, is a dominant drug determinant to the clinical indenoisoquinolines. In addition, BRCA1-, BRCA2-, and PALB2-deficient cells were hypersensitive to the indenoisoquinolines. All 3 clinical indenoisoquinolines were also synergistic with olaparib, especially in the HRD cells. The synergy between LMP400 and olaparib was confirmed in the orthotopic allograft model harboring BRCA1 loss. CONCLUSIONS: Our results provide a rationale for molecularly designed clinical trials with the indenoisoquinolines as single agents and in combination with PARP inhibitors in HRD cancers expressing SLFN11.

EGR1 regulates angiogenic and osteoclastogenic factors in prostate cancer and promotes metastasis.

Early growth response-1 (EGR1) is a transcription factor correlated with prostate cancer (PC) progression in a variety of contexts. For example, EGR1 levels increase in response to suppressed androgen receptor signaling or loss of the tumor suppressor, PTEN. EGR1 has been shown to regulate genes influencing proliferation, apoptosis, immune cell activation, and matrix degradation, among others. Despite this, the impact of EGR1 on PC metastatic colonization is unclear. We demonstrate using a PC model (DU145/RasB1) of bone and brain metastasis that EGR1 expression regulates angiogenic and osteoclastogenic properties of metastases. We have shown previously that FN14 (TNFRSF12A) and downstream NF-κB signaling is required for metastasis in this model. Here we demonstrate that FN14 ligation also leads to NF-κB-independent, MEK-dependent EGR1 expression. EGR1-depletion in DU145/RasB1 cells reduced both the number and size of metastases but did not affect primary tumor growth. Decreased EGR1 expression led to reduced blood vessel density in brain and bone metastases as well as decreased osteolytic bone lesion area and reduced numbers of osteoclasts at the bone-tumor interface. TWEAK (TNFSF12) induced several EGR1-dependent angiogenic and osteoclastogenic factors (e.g., PDGFA, TGFB1, SPP1, IL6, IL8, and TGFA, among others). Consistent with this, in clinical samples of PC, the level of several genes encoding angiogenic/osteoclastogenic pathway effectors correlated with EGR1 levels. Thus, we show here that EGR1 has a direct effect on prostate cancer metastases. EGR1 regulates angiogenic and osteoclastogenic factors, informing the underlying signaling networks that impact autonomous and microenvironmental mechanisms of cancer metastases.

CREB5 Promotes Resistance to Androgen-Receptor Antagonists and Androgen Deprivation in Prostate Cancer.

Androgen-receptor (AR) inhibitors, including enzalutamide, are used for treatment of all metastatic castration-resistant prostate cancers (mCRPCs). However, some patients develop resistance or never respond. We find that the transcription factor CREB5 confers enzalutamide resistance in an open reading frame (ORF) expression screen and in tumor xenografts. CREB5 overexpression is essential for an enzalutamide-resistant patient-derived organoid. In AR-expressing prostate cancer cells, CREB5 interactions enhance AR activity at a subset of promoters and enhancers upon enzalutamide treatment, including MYC and genes involved in the cell cycle. In mCRPC, we found recurrent amplification and overexpression of CREB5. Our observations identify CREB5 as one mechanism that drives resistance to AR antagonists in prostate cancers.

A PDX/Organoid Biobank of Advanced Prostate Cancers Captures Genomic and Phenotypic Heterogeneity for Disease Modeling and Therapeutic Screening.

Purpose: Prostate cancer translational research has been hampered by the lack of comprehensive and tractable models that represent the genomic landscape of clinical disease. Metastatic castrate-resistant prostate cancer (mCRPC) patient-derived xenografts (PDXs) recapitulate the genetic and phenotypic diversity of the disease. We sought to establish a representative, preclinical platform of PDX-derived organoids that is experimentally facile for high-throughput and mechanistic analysis.Experimental Design: Using 20 models from the LuCaP mCRPC PDX cohort, including adenocarcinoma and neuroendocrine lineages, we systematically tested >20 modifications to prostate organoid conditions. Organoids were evaluated for genomic and phenotypic stability and continued reliance on the AR signaling pathway. The utility of the platform as a genotype-dependent model of drug sensitivity was tested with olaparib and carboplatin.Results: All PDX models proliferated as organoids in culture. Greater than 50% could be continuously cultured long-term in modified conditions; however, none of the PDXs could be established long-term as organoids under previously reported conditions. In addition, the modified conditions improved the establishment of patient biopsies over current methods. The genomic heterogeneity of the PDXs was conserved in organoids. Lineage markers and transcriptomes were maintained between PDXs and organoids. Dependence on AR signaling was preserved in adenocarcinoma organoids, replicating a dominant characteristic of CRPC. Finally, we observed maximum cytotoxicity to the PARP inhibitor olaparib in BRCA2-/- organoids, similar to responses observed in patients.Conclusions: The LuCaP PDX/organoid models provide an expansive, genetically characterized platform to investigate the mechanisms of pathogenesis as well as therapeutic responses and their molecular correlates in mCRPC. Clin Cancer Res; 24(17); 4332-45. ©2018 AACR.

High-throughput screens identify HSP90 inhibitors as potent therapeutics that target inter-related growth and survival pathways in advanced prostate cancer.

The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25-30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.

Activity of durvalumab plus olaparib in metastatic castration-resistant prostate cancer in men with and without DNA damage repair mutations.

BACKGROUND: Checkpoint inhibitors have not been effective for prostate cancer as single agents. Durvalumab is a human IgG1-K monoclonal antibody that targets programmed death ligand 1 and is approved by the U.S. Food and Drug Administration for locally advanced or metastatic urothelial cancer and locally advanced, unresectable stage 3 non-small cell lung cancer. Olaparib, a poly (ADP-ribose) polymerase inhibitor, has demonstrated an improvement in median progression-free survival (PFS) in select patients with metastatic castration-resistant prostate cancer (mCRPC). Data from other trials suggest there may be improved activity in men with DNA damage repair (DDR) mutations treated with checkpoint inhibitors. This trial evaluated durvalumab and olaparib in patients with mCRPC with and without somatic or germline DDR mutations. METHODS: Eligible patients had received prior enzalutamide and/or abiraterone. Patients received durvalumab 1500 mg i.v. every 28 days and olaparib 300 mg tablets p.o. every 12 h until disease progression or unacceptable toxicity. All patients had biopsies of metastatic lesions with an evaluation for both germline and somatic mutations. RESULTS: Seventeen patients received durvalumab and olaparib. Nausea was the only nonhematologic grade 3 or 4 toxicity occurring in > 1 patient (2/17). No patients were taken off trial for toxicity. Median radiographic progression-free survival (rPFS) for all patients is 16.1 months (95% CI: 4.5-16.1 months) with a 12-month rPFS of 51.5% (95% CI: 25.7-72.3%). Activity is seen in patients with alterations in DDR genes, with a median rPFS of 16.1 months (95% CI: 7.8-18.1 months). Nine of 17 (53%) patients had a radiographic and/or PSA response. Patients with fewer peripheral myeloid-derived suppressor cells and with alterations in DDR genes were more likely to respond. Early changes in circulating tumor cell counts and in both innate and adaptive immune characteristics were associated with response. CONCLUSIONS: Durvalumab plus olaparib has acceptable toxicity, and the combination demonstrates efficacy, particularly in men with DDR abnormalities. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02484404 .

Gambogic acid inhibits thioredoxin activity and induces ROS-mediated cell death in castration-resistant prostate cancer.

Advanced prostate cancer (PrCa) is treated with androgen deprivation therapy, and although there is usually a significant initial response, recurrence arises as castrate resistant prostate cancer (CRPC). New approaches are needed to treat this genetically heterogeneous, phenotypically plastic disease. CRPC with combined homozygous alterations to PTEN and TP53 comprise about 30% of clinical samples. We screened eleven traditional Chinese medicines against a panel of androgen-independent Pten/Tp53 null PrCa-derived cell lines and identified gambogic acid (GA) as a highly potent growth inhibitor. Mechanistic analyses revealed that GA disrupted cellular redox homeostasis, observed as elevated reactive oxygen species (ROS), leading to apoptotic and ferroptotic death. Consistent with this, we determined that GA inhibited thioredoxin, a necessary component of cellular anti-oxidative, protein-reducing activity. In other clinically relevant models, GA displayed submicromolar, growth inhibitory activity against a number of genomically-representative, CRPC patient derived xenograft organoid cultures. Inhibition of ROS with N-acetyl-cysteine partially reversed growth inhibition in CRPC organoids, demonstrating ROS imbalance and implying that GA may have additional mechanisms of action. These data suggest that redox imbalances initiated by GA may be useful, especially in combination therapies, for treating the heterogeneity and plasticity that contributes to the therapeutic resistance of CRPC.

Genome-wide analysis using ChIP to identify isoform-specific gene targets.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3). The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me3. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.