Publications

2021

Malone CF, Dharia NV, Kugener G, Forman AB, Rothberg MV, Abdusamad M, Gonzalez A, Kuljanin M, Robichaud AL, Conway AS, et al.

Selective modulation of a pan-essential protein as a therapeutic strategy in cancer

. Cancer Discov. 2021.

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 employ 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 principle 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.

Ferraro GB, Ali A, Luengo A, Kodack DP, Deik A, Abbott KL, Bezwada D, Blanc L, Prideaux B, Jin X, et al.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS

. Nat Cancer. 2021;2(4):414-428.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Daria NV, Kugener G, Guenther LM, Malone CF, Durbin AD, Hong AL, Howard TP, Bandopadhayay P, Wechsler CS, Fung I, et al. A first-generation pediatric cancer dependency map. Nature Genetics. 2021;53(4):529-538.

Exciting therapeutic targets are emerging from CRISPR-based screens of high mutational-burden adult cancers. A key question, however, is whether functional genomic approaches will yield new targets in pediatric cancers, known for remarkably few mutations, which often encode proteins considered challenging drug targets. To address this, we created a first-generation pediatric cancer dependency map representing 13 pediatric solid and brain tumor types. Eighty-two pediatric cancer cell lines were subjected to genome-scale CRISPR-Cas9 loss-of-function screening to identify genes required for cell survival. In contrast to the finding that pediatric cancers harbor fewer somatic mutations, we found a similar complexity of genetic dependencies in pediatric cancer cell lines compared to that in adult models. Findings from the pediatric cancer dependency map provide preclinical support for ongoing precision medicine clinical trials. The vulnerabilities observed in pediatric cancers were often distinct from those in adult cancer, indicating that repurposing adult oncology drugs will be insufficient to address childhood cancers.

Prensner JR, Enache OM, Luria, V, Krug, K, Clauser KR, Dempster JM, Karger, A, Wang, L, Stumbraite, K, Wang, L VM, et al. Noncanonical open reading frames encode functional proteins essential for cancer cell survival. Nature Biotechnology. 2021.
Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.
Cohen-Sharir, Y, McFarland JM, Abdusamad, M, Marquis, C, Bernhard SV, Kazachkova, M, Tang, H, Ippolito MR, Laue, K, Zerbib, J, et al. Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition. Nature. 2021;590(7846):486-491.
Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

2020

Neggers JE, Paolella BR, Asfaw A, Rothberg MV, Skipper TA, Yang A, Kalekar RL, Krill-Burger JM, Dharia NV, Kugener G, et al. Correction Synthetic Lethal Interaction between the ESCRTParalog Enzymes VPS4A and VPS4B in CancersHarboring Loss of Chromosome 18q or 16q. 2020. p. 109367.

n the originally published version of this paper, the CHMP4B sgRNA sequence listed in the STAR Methods section was incorrect. Theincorrect sequence was 50-TCGATGGCACAAGCCATGAA, which is an sgRNA designed to targetCHMP2A. The correctCHMP4B-targeting sgRNA sequence that was used in the experiments for this paper is 50- TATCAACCATCGAGTTCCAG. This change does notaffect the data or conclusions of the study and now appears in the paper online.

The authors apologize for any inconvenience this error may have caused.

 
McFarland JM, Paolella BR, Warren A, Geiger-Schuller K, Shibue T, Rothberg M, Kuksenko O, Colgan WN, Jones A, Chambers E, et al. Multiplexed single-cell transcriptional response profiling to define cancer vulnerabilities and therapeutic mechanism of action. Nat Commun. 2020;11:4296.

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.

Parsons HA, Rhoades J, Reed SC, Gydush G, Ram P, Exman P, Xiong K, Lo CC, Li T, Fleharty M, et al. Sensitive Detection of Minimal Residual Disease in Patients Treated for Early-Stage Breast Cancer. Clin Cancer Res. 2020;26:2556-2564.

PURPOSE: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing. EXPERIMENTAL DESIGN: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples. RESULTS: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2-346). Clinical sensitivity was 81% (n = 13/16) in newly diagnosed MBC, 23% (n = 7/30) at postoperative and 19% (n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3-58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4-39.2 months). CONCLUSIONS: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.

Slabicki M, Kozicka Z, Petzold G, Li YD, Manojkumar M, Bunker RD, Donovan KA, Sievers QL, Koeppel J, Suchyta D, et al. The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K. Nature. 2020;585:293-297.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation(1). Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets(2). They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines(3-5), we identify CR8-a cyclin-dependent kinase (CDK) inhibitor(6)-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.