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CRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype., CRISPR-based knockout screens in cancer cells have suggested the existence of proliferation suppressor genes (PSG). Here, the authors develop an approach to systematically identify them, and reveal a PSG module involved in fatty acid synthesis and tumour suppression in acute myeloid leukemia cell lines.

Previous research has established a pricing disparity of consumer goods and services by gender such that women pay more for the same products and services than men, the so-called pink tax. This study expands this research by examining whether these price disparities persist across a wider range of personal care products including lotions, deodorants, shaving gels/creams, razors, razor cartridges, body sprays, bar soaps, liquid soaps, and shampoos. Using the largest sample of these products to date, we examined over 3000 products to test gendered pricing across these products. Our findings suggest that gender-pricing is not pervasive across products or consistently punitive toward women. Women pay more for deodorants/antiperspirants and lotions than men, while men tend to pay more for shaving creams/gels than women. Our analysis reveals another, perhaps more insidious, process at work however: the proliferation of gender differentiation for products that makes price-comparisons for the average consumer difficult. Both gender differentiation and the pink tax are founded on essentialist thinking about gender, and further reify gender structures and inequality in society.

Although PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, only a subset of patients (10-30%) achieve durable responses. With the goal of improving response and application to PD-1/PD-L1 therapies, we have leveraged our proprietary Xilio Advanced Cytokine Therapies (X-ACT) platform to develop PD1/IL2-ACT, a PD-1 blocker enhanced with a tumor-activated, engineered IL-2 agonist. The activity of IL-2 is blocked by a protein domain that prevents IL-2Rβγ binding until activated in the tumor microenvironment by matrix metalloproteinases . Tumor selective activation of IL-2 enables dosing of PD1/IL2-ACT at sufficient exposure levels to block immunosuppressive PD1/PD-L1 signaling as well as mitigating systemic IL-2 driven toxicity. PD1/IL2-ACT is designed to enable in cis engagement of IL-2 receptors on antigen experienced PD-1+ CD8+ T cells in the tumor microenvironment (TME), inducing effector T cell functions while preventing regulatory T cell (Treg) mediated immune suppression. In a preclinical model, PD1/IL2-ACT, compared to anti-PD1 alone, induced significant tumor growth inhibition. Further, PD1/IL2-ACT was better tolerated than a PD-1 directed unmasked IL-2Rβγ biased agonist in the same preclinical model. Pharmacodynamic studies showed that PD1/IL2-ACT did not significantly change immune cell profiles in the periphery but increased antigen specific CD8+ T cells in the TME, consistent with preferential engagement of PD1+ effector T cells at the tumor site. Ex vivo protease cleavage assays performed in human tumors demonstrated significant activation of PD1/IL2-ACT by tumors of multiple indications but minimal activation in human plasma. A pharmacokinetic study in nonhuman primates revealed PD1/IL2-ACT can achieve comparable exposure levels to existing PD-1 blocking immunotherapies. Taken together, these data illustrate that PD1/IL2-ACT has the potential to broaden the activity of existing immunotherapies by inhibiting the PD1/PDL1 axis as well as directing an immunostimulatory cytokine to antigen specific T cells in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Dheeraj S. Tomar, Parker Johnson, Stephanie Hsiao, Lisa Quinn, Nancy Chan, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Hanumantha R. Madala, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Sean Yang, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jijun Dong, Jennifer O'Neil, Carl U. Bialucha. Tumor-activated PD1-directed IL-2 increased antigen specific T cells in tumors and demonstrated anti-tumor activity in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 572.

Interleukin-12 (IL-12) is a proinflammatory cytokine, which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promotes cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12-based treatments has been limited by severe systemic toxicities in the clinical setting. To overcome toxicity and potentially improve the therapeutic index of IL-12 in a clinical setting, XTX301 was engineered as a half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 in circulation and non-tumor tissue, while enabling generation of an active IL-12 moiety upon cleavage of a linker sequence by matrix metalloproteinases that are enriched in the tumor microenvironment. To confirm the ability of human tumor associated proteases to activate XTX301, cleavage was assessed in primary human tumor samples and in plasma from cancer patients. We observed cleavage of XTX301 in the majority of human tumors samples tested, but no activation was observed in plasma from cancer patients. Human IL-12 does not bind and signal through the mouse IL-12 receptors, hence three murine surrogates were created for in vivo studies: mXTX301, a non-cleavable control, and an unmasked control molecule. Tumor growth inhibition was observed after a single dose of mXTX301 as low as 0.039 mg/kg. The non-cleavable form of mXTX301 was less potent than mXTX301, demonstrating that the anti-tumor activity of mXTX301 is dependent on protease activation. The unmasked control was not well tolerated, with a > 20% body weight loss observed by Day 6, resulting in 75% of animals being euthanized by Day 11. Unlike the unmasked control, mXTX301 was well-tolerated at all tested doses, as evidenced by no loss in animals’ body weights, and demonstrated minimal pharmacodynamic activity in non-tumor tissues. In tumors, mXTX301 stimulated the infiltration of cytotoxic CD8+ T cells and induced the expression of several immune-related genes including those associated with IFN-γ cell signaling, antigen processing and presentation, and defense response as well as enrichment of gene signatures for T cells, natural killer (NK) cells, macrophages and dendritic cells as determined by RNA sequencing. In non-human primates, the highest non-severely toxic dose of XTX301 was 2mg/kg dosed weekly for a total of 4 doses. In summary, a half-life extended tumor-activated IL-12 molecule, mXTX301, demonstrated anti-tumor activity in preclinical mouse models with improved tolerability compared to a systemically active IL-12 molecule suggesting that XTX301 has potential for exerting potent anti-tumor activity while widening the therapeutic index of IL-12 treatment. Citation Format: Natalia Malkova, Ekta Patel, Sallyann Vu, Damiano Fantini, Rebekah O'Donnell, Manoussa Fanny, Justin Greene, Wilson Guzman, David Crowe, Stephanie Hsiao, Parker Johnson, Megan McLaughlin, Oleg Yerov, Kurt Jenkins, Katarina Halpin-Veszeleiova, Hanumantha Rao Madala, Caitlin O'Toole, Jake Taylor, Magali Pederzoli-Ribeil, Benjamin Nicholson, Carl Uli Bialucha, Jennifer E. O'Neil. A half-life extended, tumor-activated IL-12 increased the infiltration of effector immune cells into the tumor microenvironment and demonstrated anti-tumor activity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 587.

Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including POLI and CDK4. As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.

Patients with primary biliary cholangitis (PBC) often suffer with pruritus. We describe the impact of pruritus on quality of life and how it is managed in a real-world cohort.TARGET-PBC is a longitudinal observational cohort of patients with PBC across the USA. Data include information from medical records for three years prior to the date of consent up to 5 years of follow-up. Enrolled patients were asked to complete patient-reported outcome surveys: PBC-40, 5-D itch, and the PROMIS fatigue survey. Kruskal-Wallis tests were used to compare differences in symptoms between groups.A total of 211 patients with completed PRO surveys were included in the current study. PRO respondents were compared with non-respondents in the TARGET-PBC population and were broadly similar. Pruritus was reported in 170 patients (81%), with those reporting clinically significant pruritus (30%) scoring worse across each domain of the PBC-40 and 5-D itch, more frequently having cirrhosis, and having significantly greater levels of fatigue. Patients reporting clinically significant pruritus were more likely to receive treatment, but 33% had never received treatment (no itch = 43.9%, mild itch = 38.3%).The prevalence of pruritus was high in this population, and those reporting clinically significant pruritus had a higher likelihood of having advanced disease and worse quality of life. However, this study found that pruritus in PBC is under-treated. This may be due in part to ineffectiveness of current treatments, poor tolerance, or the lack of FDA-approved medications for pruritus.

Evaluation generates critical evidence about the effectiveness of health-focused climate adaptation, who receives what benefits, and how to improve program quality. However, using evaluation to improve climate adaptation outcomes with timeliness and context-specificity is uniquely challenging. The United States Centers for Disease Control and Prevention supports health departments to implement adaptation initiatives through the Climate-Ready States and Cities Initiative (CRSCI) grant and minimize negative health impacts of climate change via the Building Resilience Against Climate Effects (BRACE) framework, which includes evaluation. In this paper, we present current evaluation practice by describing the health-focused adaptation actions that were evaluated among CRSCI recipients, the evaluation approaches they used, and the barriers and enablers encountered. Using a mixed methods approach, we abstracted annual progress report and standardized performance measure data to characterize evaluation activities across 18 grant recipients using basic quantitative descriptive analysis. Through structured interviews with three grant recipients and standard team-based qualitative coding and analysis techniques, we developed qualitative case studies to explore evaluation barriers and enablers in richer context. Recipients reported 76 evaluations over the reporting period (2018–2021). Evaluated programs commonly addressed extreme weather (50.0%), followed by heat (27.6%). The most common purpose was to monitor implementation or improve delivery (57.9%). Case studies highlighted barriers to successful evaluation such as limited specialized evaluation expertise and staff time. Enablers included staff motivation to justify program expansion, strong relationships with community partners, and use of evaluation plans. Case studies revealed diverse strategies to seek input from stakeholders disproportionately impacted by climate change and to reduce health disparities. The experiences of CDC grant recipients provide an opportunity to better understand the barriers and enablers of climate and health evaluation practice and to identify practical strategies to expand the value of evaluation in this nascent field.

A 50-year-old male smoker with hypertension presented with subacute bilateral leg swelling, weight gain, and dyspnea. Exam revealed tachycardia, jugular venous distention, peripheral edema, diminished breath sounds, and pulsus paradoxus. Labs were notable for elevated creatinine and leukocytosis. ECG showed sinus tachycardia and low voltages. CXR showed left hemithorax opacification. TTE revealed normal LV function, septal flattening, severely dilated RV with reduced function, RVSP 81 mmHg, and a large pericardial effusion with exaggerated respirophasic variation in mitral inflow but no chamber collapse. The elevated RVSP with RV failure raised concern for acute pulmonary embolism (PE). CT chest excluded PE but revealed a large mediastinal mass compressing both pulmonary arteries (PA) and large left pleural effusion. Pulmonary angiogram demonstrated a totally occluded left PA and severe stenosis of the right PA. Right heart catheterization showed RAP 24, RV 83/21, PA 84/29 (mean 53), intrapericardial pressure 15 mmHg, and low cardiac index (1.6 L/min/m2). Pericardiocentesis was performed with close monitoring by PA catheter. Removal of 500cc fluid was well tolerated with stable hemodynamics, cardiac output, and intracardiac pressures. Pericardial fluid cytology and lymph node biopsy demonstrated small cell lung cancer. Cautious diuresis was initiated. After one cycle of chemotherapy, repeat CT chest showed reduced extrinsic compression of the right PA with improved lung perfusion. Repeat TTE showed dramatic improvement with normal RV size and function, RVSP 22 mmHg, and trivial pericardial effusion. Pericardial effusion in the setting of severe PH is associated with poor outcomes and presents both diagnostic and therapeutic challenges. Classic findings of tamponade (e.g. right sided chamber collapse) may be masked by elevated right heart pressures. Compression of the PA by a mediastinal mass is an unusual cause of PH and right heart failure that can mimic acute PE. This rare clinical scenario presents the following challenges: (1) diuresis may precipitate tamponade, (2) pericardiocentesis may enable an already enlarged RV to acutely dilate and thus worsen RV failure, and (3) there is uncertainty regarding the role for PA stenting.

BackgroundInterleukin-12 (IL-12) is a proinflammatory cytokine which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promoting cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12 has been limited by severe systemic toxicities. To overcome toxicity and improve the therapeutic index of IL-12, we employed protein engineering to generate XTX301, a highly potent, half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 systemically and render an active IL-12 moiety upon cleavage by proteases that are enriched in the tumor microenvironment.MethodsWe conducted experiments to assess the binding, bioactivity, safety, and anti-tumor efficacy of XTX301. Binding interactions were measured via SPR, bioactivity was measured using STAT-4 phosphorylation in a reporter cell line, and IFN-g production was assessed in human PBMCs via ELISA. Anti-tumor efficacy and pharmacodynamics were assessed in MC38 and B16F10 syngeneic tumor mouse models using a XTX301 murine surrogate, mXTX301. Safety and pharmacokinetics of XTX301 were evaluated in non-human primates (NHP).ResultsXTX301 showed no detectable binding to the high affinity IL12RB2 demonstrating that the masking domain indeed prevents interaction with the receptor. Upon cleavage of the masking domain by relevant proteases, binding was observed and was comparable to XTX300 unmasked control. Likewise, restoration of activity upon proteolytic cleavage was observed in an IL-12-dependent reporter gene assay and in primary human PBMCs. Human IL-12 does not cross react with mouse IL-12 receptors; hence a murine surrogate (mXTX301) was created for in vivo anti-tumor efficacy evaluation. A single dose of mXTX301 demonstrated up to 90% tumor growth inhibition in an inflamed MC38 and non-inflamed B16F10 syngeneic mouse models. mXTX301 induced a ~3 fold increase in IFN-g in tumors compared to vehicle control and ~150 fold less peripheral IFN-g compared to mXTX300. XTX301 exhibits minimal elevation in liver enzymes and a 50-fold improvement in tolerability compared to XTX300, in a repeat dose NHP safety study.ConclusionsOur data demonstrates that both XTX301 and mXTX301 are inactive when in masked form and become activated upon proteolytic cleavage to exert bioactivity comparable to recombinant IL-12. For efficacy, mXTX301 demonstrated tumor selective activity in syngeneic mouse models. XTX301 was well tolerated in repeat dose NHP safety study. In conclusion, XTX301 has potential for exerting potent anti-tumor activity with a favorable tolerability profile.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin β6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin β6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

PDZ domains are key players in signalling pathways. These modular domains generally recognize short linear C-terminal stretches of sequences in proteins that organize the formation of complex multi-component assemblies. The development of new methodologies for the characterization of the molecular principles governing these interactions is critical to fully understand the functional diversity of the family and to elucidate biological functions for family members. Here, we applied an in vitro evolution strategy to explore comprehensively the capacity of PDZ domains for specific recognition of different amino acids at a key position in C-terminal peptide ligands. We constructed a phage-displayed library of the Erbin PDZ domain by randomizing the binding site

State and local public health agencies are at the forefront of planning and responding to the health challenges of climate hazards but face substantial barriers to effective climate and health adaptation amidst concurrent environmental and public health crises. To ensure successful adaptation, it is necessary to understand and overcome these barriers. The U.S. Centers for Disease Control and Prevention Climate-Ready States and Cities Initiative (CRSCI) provides funding to state and local health departments to anticipate and respond to health impacts from climate change using the Building Resilience Against Climate Effects (BRACE) framework. This paper explores the barriers to and enablers of successful adaptation projects among BRACE West CRSCI grantees, including Arizona, California, Oregon, and the city and county of San Francisco. The barriers included competing demands such as the COVID-19 pandemic, dependence on partners with similar challenges, staff and leadership turnover, uncertain and complex impacts on at-risk populations, and inadequate resources. The enablers included effective partnerships, leadership support, dedicated and skilled internal staff, and policy windows enabling institutional change and reprioritization. These findings highlight effective strategies in the field that state and local health departments may use to anticipate potential barriers and establish their work in an environment conducive to successful adaptation.

Background High-dose recombinant human interleukin-2 (aldesleukin) elicits durable anti-tumor immunity and gained FDA approval two decades prior to checkpoint blockers. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. Second generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a 2nd generation IL-2 surrogate that does not bind IL-2Rα still experience characteristic dose-limiting toxicities, including vascular leak syndrome (VLS), and exhibit dysregulated peripheral immune function due to reduced Treg activation. To overcome these toxicities and improve the therapeutic index of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX201, a highly potent 3rd generation IL-2 that is designed to be selectively active in tumors, stimulating cytolytic responses against tumor cells while sparing systemic immune activation. Methods XTX201 binding interactions were measured with SPR, and bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and immune activation was evaluated in tumors compared to peripheral organs in syngeneic tumor mouse models. Safety and pharmacokinetics were evaluated in rodents and NHPs. Results Non-activated XTX201 showed no detectable binding to IL-2Rα or IL-2Rβ, and limited IL-2R-dependent STAT-5 signaling in vitro. Activation of XTX201 resulted in high-affinity binding to IL-2Rβ and no binding to IL-2Rα, leading to a ~1000-fold reduction in Treg activation as compared to WT IL-2, while retaining CD8+ T and NK cell activation. Mice and NHPs treated with a 2nd generation IL-2 surrogate experienced toxicities that are commonly observed in patients treated with aldesleukin, including pulmonary edema, VLS, fever and lethality. However, XTX201 did not induce toxicities at exposures 100-fold higher than the MTD of the activated version, and achieved similar anti-tumor efficacy in mice. Experiments in primary human solid tumors and human plasma indicated that XTX201 is preferentially activated in the tumor microenvironment. Conclusions Our data demonstrate that 2nd generation IL-2s that are systemically active and lack binding to IL-2Rα exhibit dose-limiting toxicities unless further engineered for selective activity in tumors. XTX201, a 3rd generation, tumor-selective IL-2, exhibits a long half-life and is innocuous outside of tumors. XTX201 is activated within tumors to release an IL-2Rβ/γ biased cytokine that inhibits tumor growth in syngeneic models, and exhibits tumor-specific pharmacodynamic effects without peripheral toxicities. XTX201 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of aldesleukin while minimizing dose-limiting toxicities.

BackgroundPooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell.ResultsThrough a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes.ConclusionsTogether, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.

CRISPR knockout screens in hundreds of cancer cell lines have revealed a substantial number of context-specific essential genes that, when associated with a biomarker such as lineage or oncogenic mutation, offer candidate tumor-specific vulnerabilities for targeted therapies or novel drug development. Data-driven analysis of knockout fitness screens also yields many other functionally coherent modules that show emergent essentiality or, in rarer cases, the opposite phenotype of faster proliferation. We develop a systematic approach to classify these suppressors of proliferation, which are highly enriched for tumor suppressor genes, and define a network of 145 genes in 22 discrete modules. One surprising module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of AML lines, which we call Fatty Acid Synthesis/Tumor Suppressor (FASTS) cells. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe fitness phenotype for the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirmed biochemically. Overexpression of genes in this module is associated with a survival advantage in an age-matched cohort of AML patients, suggesting the gene cluster driving an in vitro phenotype may be associated with a novel, clinically relevant subtype.

Major efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.