WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Nanoliposome targeting in breast cancer is influenced by the tumor microenvironment.

MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.

Rare somatic cells from human breast tissue exhibit extensive lineage plasticity.

We identified cell surface markers associated with repression of p16(INK4a)/cyclin-dependent kinase inhibitor 2A(CDKN2A), a critical determinant in the acquisition of a plastic state. These cell surface markers allowed direct isolation of rare cells from healthy human breast tissue that exhibit extensive lineage plasticity. This subpopulation is poised to transcribe plasticity markers, OCT3/4, SOX2, and NANOG, at levels similar to those measured in human embryonic stem cells and to acquire a plastic state sensitive to environmental programming. In vitro, in vivo, and teratoma assays demonstrated that either a directly sorted (uncultured) or a single-cell (clonogenic) cell population from primary tissue can differentiate into functional derivatives of each germ layer, ectodermal, endodermal, and mesodermal. In contrast to other cells that express OCT3/4, SOX2, and NANOG, these human endogenous plastic somatic cells are mortal, express low telomerase activity, expand for an extensive but finite number of population doublings, and maintain a diploid karyotype before arresting in G1.

Identification of Small Molecule Inhibitors of PPM1D Using a Novel Drug Discovery Platform.

Protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D), is a serine/threonine phosphatase that is recurrently activated in cancer, regulates the DNA damage response (DDR), and suppresses the activation of p53. Consistent with its oncogenic properties, genetic loss or pharmacologic inhibition of PPM1D impairs tumor growth and sensitizes cancer cells to cytotoxic therapies in a wide range of preclinical models. Given the therapeutic potential of targeting PPM1D specifically and the DDR and p53 pathway more generally, we sought to deepen our biological understanding of PPM1D as a drug target and determine how PPM1D inhibition differs from other therapeutic approaches to activate the DDR. We performed a high throughput screen to identify new allosteric inhibitors of PPM1D, then generated and optimized a suite of enzymatic, cell-based, and in vivo pharmacokinetic and pharmacodynamic assays to drive medicinal chemistry efforts and to further interrogate the biology of PPM1D. Importantly, this drug discovery platform can be readily adapted to broadly study the DDR and p53. We identified compounds distinct from previously reported allosteric inhibitors and showed in vivo on-target activity. Our data suggest that the biological effects of inhibiting PPM1D are distinct from inhibitors of the MDM2-p53 interaction and standard cytotoxic chemotherapies. These differences also highlight the potential therapeutic contexts in which targeting PPM1D would be most valuable. Therefore, our studies have identified a series of new PPM1D inhibitors, generated a suite of in vitro and in vivo assays that can be broadly used to interrogate the DDR, and provided important new insights into PPM1D as a drug target.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

No paradox, no progress: inverse cancer comorbidity in people with other complex diseases.

In the past 5 years, several leading groups have attempted to explain why individuals with Down's syndrome have a reduced risk of many solid tumours and an increased risk of leukaemia and testicular cancer. Niels Bohr, the Danish physicist, noted that a paradox could initiate progress. We think that the paradox of a medical disorder protecting against cancer could be formalised in a new model of inverse cancer morbidity in people with other serious diseases. In this Personal View, we review evidence from epidemiological and clinical studies that supports a consistently lower than expected occurrence of cancer in patients with Down's syndrome, Parkinson's disease, schizophrenia, diabetes, Alzheimer's disease, multiple sclerosis, and anorexia nervosa. Intriguingly, most comorbidities are neuropsychiatric or CNS disorders. We provide a brief overview of evidence indicating genetic and molecular connections between cancer and these complex diseases. Inverse comorbidity could be a valuable model to investigate common or related pathways or processes and test new therapies, but, most importantly, to understand why certain people are protected from the malignancy.

Phosphate dysregulation via the XPR1-KIDINS220 protein complex is a therapeutic vulnerability in ovarian cancer.

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic "vacuolar" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

Sustained induction of epithelial to mesenchymal transition activates DNA methylation of genes silenced in basal-like breast cancers.

The active acquisition of epigenetic changes is a poorly understood but important process in development, differentiation, and disease. Our work has shown that repression of the p16/pRb pathway in human epithelial cells, a condition common to stem cells and many tumor cells, induces dynamic epigenetic remodeling resulting in the targeted methylation of a selected group of CpG islands. We hypothesized that cells in this epigenetically plastic state could be programmed by the microenvironment to acquire epigenetic changes associated with tumorigenesis. Here, we describe an in vitro model system where epigenetically plastic cells were placed in an environment that induced epithelial to mesenchymal transition (EMT) and led to a program of acquired de novo DNA methylation at targeted sites. In this model, we found that repression of E-cadherin transcription preceded the subsequent acquisition of methylated CpG sites. Furthermore, the induction of EMT was accompanied by de novo methylation of several other gene promoters, including those of the estrogen receptor and Twist. These data demonstrate that signals from the microenvironment can induce phenotypic and gene expression changes associated with targeted de novo epigenetic alterations important in tumor progression, and that these alterations occur through a deterministic, rather than stochastic, mechanism. Given the dynamic epigenetic reprogramming that occurs in these cells, DNA methylation profiles observed in human tumors may reflect the history of environmental exposures during the genesis of a tumor.

Selective Modulation of a Pan-Essential Protein as a Therapeutic Strategy in Cancer.

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity. SIGNIFICANCE: We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1-NXT2 paralog relationship.See related commentary by Wang and Abdel-Wahab, p. 2129.This article is highlighted in the In This Issue feature, p. 2113.

Synthetic Lethal Interaction between the ESCRT Paralog Enzymes VPS4A and VPS4B in Cancers Harboring Loss of Chromosome 18q or 16q.

Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.

Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling.

Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy.

INTRODUCTION: We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. METHODS: HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). RESULTS: vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. CONCLUSIONS: We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases.

TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed, oncogene-transformed, and human cancer cells.

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.

Dual role of transforming growth factor beta in mammary tumorigenesis and metastatic progression.

It is generally accepted that transforming growth factor beta (TGFbeta) is both a tumor suppressor and tumor promoter. Whereas loss or attenuation of TGFbeta signal transduction is permissive for transformation, introduction of dominant-negative TGFbeta receptors into metastatic breast cancer cells has been shown to inhibit epithelial-to-mesenchymal transition, motility, invasiveness, survival, and metastases. In addition, there is evidence that excess production and/or activation of TGFbeta by cancer cells can contribute to tumor progression by paracrine mechanisms involving neoangiogenesis, production of stroma and proteases, and subversion of immune surveillance mechanisms in tumor hosts. These data provide a rationale in favor of blockade of autocrine/paracrine TGFbeta signaling in human mammary tumors with therapeutic intent. Several treatment approaches are currently in early clinical development and have been the focus of our laboratory. These include (1) ligand antibodies or receptor-containing fusion proteins aimed at blocking ligand binding to cognate receptors and (2) small-molecule inhibitors of the type I TGFbeta receptor serine/threonine kinase. Many questions remain about the viability of anti-TGFbeta treatment strategies, the best molecular approach (or combinations) for inhibition of TGFbeta function in vivo, the biochemical surrogate markers of tumor response, the molecular profiles in tumors for selection into clinical trials, and potential toxicities, among others.

Cyclophosphamide-Mediated Tumor Priming for Enhanced Delivery and Antitumor Activity of HER2-Targeted Liposomal Doxorubicin (MM-302).

Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer.