Paradoxical activation of oncogenic signaling as a cancer treatment strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.

cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis.

Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this 'one-two punch' cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Genetic and compound screens uncover factors modulating cancer cell response to indisulam.

Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4DCAF15 Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Last, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

The Cancer SENESCopedia: A delineation of cancer cell senescence.

Cellular senescence is characterized as a stable proliferation arrest that can be triggered by multiple stresses. Most knowledge about senescent cells is obtained from studies in primary cells. However, senescence features may be different in cancer cells, since the pathways that are involved in senescence induction are often deregulated in cancer. We report here a comprehensive analysis of the transcriptome and senolytic responses in a panel of 13 cancer cell lines rendered senescent by two distinct compounds. We show that in cancer cells, the response to senolytic agents and the composition of the senescence-associated secretory phenotype are more influenced by the cell of origin than by the senescence trigger. Using machine learning, we establish the SENCAN gene expression classifier for the detection of senescence in cancer cell samples. The expression profiles and senescence classifier are available as an interactive online Cancer SENESCopedia.

Identification of Autophagy-Related Genes as Targets for Senescence Induction Using a Customizable CRISPR-Based Suicide Switch Screen.

Pro-senescence therapies are increasingly being considered for the treatment of cancer. Identifying additional targets to induce senescence in cancer cells could further enable such therapies. However, screening for targets whose suppression induces senescence on a genome-wide scale is challenging, as senescent cells become growth arrested, and senescence-associated features can take 1 to 2 weeks to develop. For a screen with a whole-genome CRISPR library, this would result in billions of undesirable proliferating cells by the time the senescent features emerge in the growth arrested cells. Here, we present a suicide switch system that allows genome-wide CRISPR screening in growth-arrested subpopulations by eliminating the proliferating cells during the screen through activation of a suicide switch in proliferating cells. Using this system, we identify in a genome-scale CRISPR screen several autophagy-related proteins as targets for senescence induction. We show that inhibiting macroautophagy with a small molecule ULK1 inhibitor can induce senescence in cancer cell lines of different origin. Finally, we show that combining ULK1 inhibition with the senolytic drug ABT-263 leads to apoptosis in a panel of cancer cell lines. IMPLICATIONS: Our suicide switch approach allows for genome-scale identification of pro-senescence targets, and can be adapted to simplify other screens depending on the nature of the promoter used to drive the switch.

Fighting Drug Resistance through the Targeting of Drug-Tolerant Persister Cells.

Designing specific therapies for drug-resistant cancers is arguably the ultimate challenge in cancer therapy. While much emphasis has been put on the study of genetic alterations that give rise to drug resistance, much less is known about the non-genetic adaptation mechanisms that operate during the early stages of drug resistance development. Drug-tolerant persister cells have been suggested to be key players in this process. These cells are thought to have undergone non-genetic adaptations that enable survival in the presence of a drug, from which full-blown resistant cells may emerge. Such initial adaptations often involve engagement of stress response programs to maintain cancer cell viability. In this review, we discuss the nature of drug-tolerant cancer phenotypes, as well as the non-genetic adaptations involved. We also discuss how malignant cells employ homeostatic stress response pathways to mitigate the intrinsic costs of such adaptations. Lastly, we discuss which vulnerabilities are introduced by these adaptations and how these might be exploited therapeutically.

A Novel Platform to Test In Vivo Single Gene Dependencies in t(8,21) and t(15,17) AML Confirms Zeb2 as Leukemia Target.

The increased usage of high-throughput technologies in cancer research, including genetic and drug screens, generates large sets of candidate targets that need to be functionally validated for their roles in tumor development. Thus, reliable and robust in vivo model systems are needed to perform reverse genetic experiments. Ideally, these models should allow for a conditional silencing of the target and an unambiguous identification of engineered cancer cells. Here, we present a platform consisting of: (i) t(8;21) and t(15;17) driven acute myeloid leukemia (AML) transgenic mice with constitutive expression of green fluorescent protein (GFP) and inducible expression of Cre recombinase, and (ii) REX, a modified pSico lentiviral vector for inducible shRNA expression and red fluorescent protein (RFP) as a selection marker. In this system, leukemic cells from transgenic mice are transduced with REX, flow sorted, and transplanted into syngeneic hosts. Gene interference is induced in established tumors by tamoxifen treatment. Dual-color cell fluorescence guides the in vivo identification of shRNA interfered AML cells, monitoring engraftment and disease progression. We tested the platform by inducing knockdown of Zeb2, a gene upregulated by AML1-ETO and PML-RARα oncogenes in pre-leukemic hematopoietic stem cell compartment, and observed a significant delay in leukemia onset. This proves the power and utility of the platform and confirms Zeb2 contribution to the pathogenesis of AML.

Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations.

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.