Supplementary Figures S1-S8 with legends.

In this paper, we study the generalized gapped k-mer filters and derive a closed form solution for their coefficients. We consider nonnegative integers $\ell$ and $k$, with $k\leq \ell$, and an $\ell$-tuple $B=(b_1,\ldots,b_{\ell})$ of integers $b_i\geq 2$, $i=1,\ldots,\ell$. We introduce and study an incidence matrix $A=A_{\ell,k;B}$. We develop a M\"obius-like function $\nu_B$ which helps us to obtain closed forms for a complete set of mutually orthogonal eigenvectors of $A^{\top} A$ as well as a complete set of mutually orthogonal eigenvectors of $AA^{\top}$ corresponding to nonzero eigenvalues. The reduced singular value decomposition of $A$ and combinatorial interpretations for the nullity and rank of $A$, are among the consequences of this approach. We then combine the obtained formulas, some results from linear algebra, and combinatorial identities of elementary symmetric functions and $\nu_B$, to provide the entries of the Moore-Penrose pseudo-inverse matrix $A^{+}$ and the Gapped k-mer filter matrix $A^{+} A$.

Abstract Microsatellite instability high (MSI-H) tumors are marked by 30% or more mutations in microsatellite regions and are generated by somatic or germline alterations in DNA mismatch repair machinery. Despite a high tumor mutational burden, MSI-H tumors do not frequently harbor mutations in TP53. Notably, a frameshift mutation (p.K15fs) in the gene RPL22 is among the most recurrent mutations in MSI-H tumors. RPL22 and its paralog, RPL22L1, form part of the large subunit of the 60S ribosome and are known to affect protein synthesis as well as splicing of genes and transcription factors that affect development and tumorigenesis. Here we explore the result of RPL22 loss on the regulation of MDM4, an upstream target of p53. MDM4 protein expression is mediated through alternative mRNA splicing of its sixth exon, which leads to two isoforms, the exon 6-inclusive MDM4-FL and the exon 6-exclusive MDM4-S, which is prone to degradation. To date, no mutational events have been associated with this alternative splicing event. We show the prevalence of RPL22fs mutations is more than 70% in certain MSI-H cell lines (e.g. colon, endometrial) and tumors (e.g. stomach adenocarcinoma) in the CCLE and TCGA. RPL22 copy number loss and RPL22 frameshift mutations are strongly correlated with MDM4 exon 6 inclusion in TCGA samples, and RPL22 knockout increases MDM4 exon 6 inclusion and MDM4 protein in multiple cell lines. RPL22 loss increases cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. Downstream targets of p53 (p21, BBC3) are downregulated due to RPL22 loss and Nutlin-3a inhibition, suggesting mutation of RPL22 and subsequent overexpression of MDM4 promotes resistance to Nutlin-3a. CLIP-seq revealed that RPL22 binds the MDM4 3’-UTR. We find that RPL22 loss is associated with an alternative 3’-splicing event in RPL22L1 adjacent to exon 3 and results in the expression of the full-length transcript of RPL22L1. In summary, we identify RPL22 as a key modulator of MDM4 splicing through an alternative splicing switch in exon 6. Furthermore, RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated functional transcript. Therefore, we propose that damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction. Our results also suggest that the presence of RPL22 may act as a rheostat to maintain MDM4 exon 6 skipping under normal cellular contexts. Citation Format: Hannah N. Weinstein, Kevin Hu, Lisa Fish, Yih-An Chen, Paul Allegakoen, Keliana S. Hui, Julia H. Pham, Maria B. Baco, Hanbing Song, Andrew O. Giacomelli, Francisca Vazquez, Mahmoud Ghandi3, Hani Goodarzi, Franklin W. Huang. RPL22 is a tumor suppressor in MSI-high cancers and a key splicing regulator of MDM4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1256.

Microsatellite instability-high (MSI-H) tumors are malignant tumors that, despite harboring a high mutational burden, often have intact TP53. One of the most frequent mutations in MSI-H tumors is a frameshift mutation in RPL22, a ribosomal protein. Here, we identified RPL22 as a modulator of MDM4 splicing through an alternative splicing switch in exon 6. RPL22 loss increases MDM4 exon 6 inclusion and cell proliferation and augments resistance to the MDM inhibitor Nutlin-3a. RPL22 represses the expression of its paralog, RPL22L1, by mediating the splicing of a cryptic exon corresponding to a truncated transcript. Therefore, damaging mutations in RPL22 drive oncogenic MDM4 induction and reveal a common splicing circuit in MSI-H tumors that may inform therapeutic targeting of the MDM4-p53 axis and oncogenic RPL22L1 induction.

Supplementary Methods, Tables 1 - 9, Figures 1 - 8. Supplementary Table 1. Functional classification for validated genes. Supplementary Table 2. Validated 43 candidate genes' genetic alteration status in the invasive breast cancer (TCGA) database. Supplementary Table 3. Receptor status, mutational status and intrinsic subtypes of breast cancer cell lines used in this study. Supplementary Table 4. GI50 of BYL719 in different breast cancer cell lines with overexpression of PIM isoforms. Supplementary Table 5. PIM1 and AKT phosphorylation sites in PRAS40, BAD, p21 and p27. Supplementary Table 6. PIM gene expression levels in PIK3CA-mutant breast cancer cell lines. Supplementary Table 7. GI50 of BYL719 in the presence of LGH447 (1μM) or DMSO in PIK3CA-mutant breast cancer cell lines. Supplementary Table 8. Signature genes that changed concordantly in patient sample pairs and PIM signature. Supplementary Table 9. Hormonal receptor status by immunohistochemistry (IHC), HER2 status by FISH and IHC of PIM1 amplified or overexpressed breast cancer tumor samples from the invasive breast cancer (TCGA) database. Supplementary Figure 1. Lentiviral infection efficacy quality in the large-scale ORF screen. Supplementary Figure 2. PIM1 confers resistance to breast cancer cell lines with different PIK3CA mutations and various intrinsic subtypes. Supplementary Figure 3. PIM1 confers resistance to inhibitors targeting key components of PI3K pathway. Supplementary Figure 4. PIM1 inhibitors alone have minimal inhibitory effects on T47D cells with or without PIM1 overexpression. Supplementary Figure 5. PIM1 expression abrogates cell-cycle arrest in cells treated with BYL719. Supplementary Figure 6. BYL719 and AZD1208 or LGH447 demonstrated synergy in PIM1 expressing T47D cells. Supplementary Figure 7. PIM activation signature. Supplementary Figure 8. PIM and PI3K pathway genetic mutations showed mutual exclusivity in treatment-naive breast cancers.

Supplemental Figures 1-8. Fig. S1. The landscape of mutations of putative ERK1/2 resistance alleles; Fig. S2. Expression and signaling induced by validated ERK resistance alleles; Fig. S3. ERK2 signaling and kinase activity assays with the ERK inhibitor SCH772984; Fig. S4. Analysis of co-mutated ERK1/2 alterations; Fig. S5. Structural localization of RAF/MEK inhibitor alleles; Fig. S6. Drug sensitivity of non-validating alleles; Fig. S7. Drug sensitivity assays of RAF/MEK inhibitor resistance alleles; Fig. S8. Viability of ERK1/2 resistance alleles in WM266.4 cells

Abstract MYC transcription factors have been demonstrated to be drivers of many cancers, yet have remained recalcitrant to new drug development. We hypothesized that MYC-driven cancer cell lines are addicted to protein translation, and therefore are vulnerable to the loss of the translation termination factor, GSPT1. We have discovered MRT-2359, a selective GSPT1 molecular glue degrader, that demonstrates potent and preferential antiproliferative activity in MYC-driven cell lines, such as high N- and L-MYC mRNA expressing non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) lines. MRT-2359 has been optimized for differential toxicity between MYC- and non-MYC-driven cancer cell lines, kinetics and depth of degradation, oral bioavailability, and in vivo efficacy. The anti-tumor activity of MRT-2359 was assessed in more than 80 lung patient-derived xenografts (PDXs) that confirmed the preferential anti-tumor activity in N- and L-MYC high NSCLC and SCLC PDXs when dosed orally daily or intermittently. Similar levels of activity were also observed in neuroendocrine lung cancer PDXs and lymphoma models. Oral MRT-2359 is currently in a Phase 1/2 clinical trial in selected cancer patients with MYC-driven NSCLC, SCLC, high grade neuroendocrine cancers and diffuse large B-cell lymphoma (NCT05546268). Citation Format: Owen B. Wallace, Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Maciej Cabanski, Lisa Cantagallo, Agustin Chicas, Qian Chen, Anna Diesslin, Christopher King, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Dave Peck, Carolina Perdomo Ortiz, Martin Schillo, Ambika Singh, Ralph Tiedt, Simone Tortoioli, Silvia Buonamici, Markus Warmuth, Filip Janku, Bernhard Fasching. Discovery of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for MYC-driven cancers [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 ND10.

MYC transcription factors are well-established drivers of human cancers but despite being amongst the most frequently altered oncogenes, no approved therapy targeting MYC-driven tumors has been developed to date. MYC-driven cancers are known to be addicted to protein translation. This addiction creates a dependency on critical components of the translational machinery providing in turn a unique opportunity for therapeutic intervention. We hypothesized that targeting the translation termination factor GSPT1, a key regulator of protein synthesis, would constitute a vulnerability for MYC-driven tumors. Herein we further describe MRT-2359 a potent, selective and orally bioavailable degrader of GSPT1. MRT-2359 was rationally designed using our QuEENTM discovery engine and optimized to achieve a profound and preferential antiproliferative activity in MYC-driven cell lines, such as high N- and L-MYC mRNA expressing non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) lines. In line with expectations, MRT-2359 activity is dependent on both CRBN and the GSPT1 G-loop degron. We further demonstrate using an inducible system that the sole expression of either N- or L-MYC is sufficient to sensitize initially resistant NSCLC cells to MRT-2359. These studies therefore establish a causal link between N- and L-MYC expression and sensitivity to MRT-2359. Unlike MRT-2359, agents targeting the protein translation initiation machinery or repressing MYC transcription (CDK9 inhibitor) failed to show such differential activity. Mechanistically, RiboSeq and polysome profiling revealed that treatment with MRT-2359 in the N- or L-MYC high cell lines induces ribosome stalling at the stop codon, increased monosomes and decreased polysomes. These changes are indicative of translational repression and were confirmed using puromycilation assays. Proteomics and RNAseq studies finally demonstrated a significant reduction in the total levels of N- or L-MYC leading in turn to the downmodulation of MYC target genes. Despite robust degradation of GSPT1, no marked effect was observed in these assays in low N- or L-MYC lines, confirming the selective activity of MRT-2359 in MYC-driven lung cancers. Last, the anti-tumor activity of MRT-2359 was assessed in >80 lung patient-derived xenografts (PDXs). MRT-2359 demonstrated preferential activity in N- and L-MYC high NSCLC and SCLC PDXs, including numerous instances of tumor regressions, when dosed orally daily or intermittently. Similar levels of anti-tumor activity were also observed in neuroendocrine lung cancer and lymphoma PDXs. Together these results warrant further investigations in the clinic. Oral MRT-2359 is currently in a Phase 1/2 clinical trial in selected cancer patients with MYC-driven NSCLC, SCLC, high grade neuroendocrine cancers and diffuse large B-cell lymphoma (NCT05546268). Citation Format: Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Maciej Cabanski, Lisa Cantagallo, Agustin Chicas, Qian Chen, Anna Diesslin, Christopher King, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Dave Peck, Carolina Perdomo Ortiz, Martin Schillo, Ambika Singh, Ralph Tiedt, Simone Tortoioli, Silvia Buonamici, Filip Janku, Owen Wallace, Bernhard Fasching. Development of MRT-2359, an orally bioavailable GSPT1 molecular glue degrader, for the treatment of lung cancers with MYC-induced translational addiction [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 3449.

Here, we present FusionInspector for in silico characterization and interpretation of candidate fusion tran-scripts from RNA sequencing (RNA-seq) and exploration of their sequence and expression characteristics. We applied FusionInspector to thousands of tumor and normal transcriptomes and identified statistical and experimental features enriched among biologically impactful fusions. Through clustering and machine learning, we identified large collections of fusions potentially relevant to tumor and normal biological pro-cesses. We show that biologically relevant fusions are enriched for relatively high expression of the fusion transcript, imbalanced fusion allelic ratios, and canonical splicing patterns, and are deficient in sequence mi-crohomologies between partner genes. We demonstrate that FusionInspector accurately validates fusion transcripts in silico and helps characterize numerous understudied fusions in tumor and normal tissue sam-ples. FusionInspector is freely available as open source for screening, characterization, and visualization of candidate fusions via RNA-seq, and facilitates transparent explanation and interpretation of machine -learning predictions and their experimental sources.

<p>Supplemental Text. Supplemental Materials and Methods, primer sequences, Supplemental Figure Legends, and references.</p>

Supplemental Table 1 and 2. Amino acid substitutions for each significantly mutated ERK1/2 nucleotide for the ERK inhibitor screen (Table 1) or the RAF/MEK inhibitor screen (Table 2) and the accompanying resistance phenotype.

Myc transcription factors are well-established drivers of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy targeting the Myc family members directly has been developed to date. To sustain uncontrolled cell proliferation and tumor growth, Myc-driven cancers are known to be addicted to protein translation. This addiction creates a dependency on critical components of the translational machinery providing in turn a unique opportunity for therapeutic intervention. We hypothesized that targeting the translational termination factor GSPT1, a key regulator of protein synthesis, would constitute a vulnerability for Myc-driven tumors. GSPT1 contains a well-defined degron allowing for the recruitment of the E3 ligase cereblon (CRBN) and subsequent proteasomal degradation in the presence of molecular glue degraders. Herein we describe a novel orally bioavailable GSPT1-directed small molecule degrader MRT-2359, which has been rationally designed and optimized to selectively induce apoptosis in translationally addicted cells. MRT-2359 promotes complex formation between CRBN and GSPT1 and potently induces GSPT1 degradation in a CRBN- and degron-dependent manner. The high selectivity of MRT-2359 was subsequently demonstrated by the lack of activity in cells expressing a non-degradable GSPT1 mutant. Although MRT-2359 degrades GSPT1 in all the cell lines tested, profiling in a large panel of cancer lines revealed profound and preferential antiproliferative activity in Myc-driven cell lines, such as high N-Myc expressing non-small cell lung cancer (NSCLC) lines and high L-Myc expressing small cell lung cancer (SCLC) lines. In the Myc-driven cells, degradation of GSPT1 led to translational repression as manifested by a global shift from polysomes to monosomes resulting in the reduction of a subset of proteins as assessed by quantitative proteomics. In particular, N- or L-Myc protein levels decreased and as a consequence the known Myc target genes were downregulated at the mRNA level. Despite the robust degradation of GSPT1, no marked effect was observed in low N-Myc lines, confirming the selective activity of our GSPT1 degrader in Myc-driven lung cancers. Finally, oral administration of MRT-2359 in high N-Myc NSCLC xenografts and PDXs led to complete intratumoral GSPT1 degradation and concomitant decrease in N-Myc protein levels, resulting in tumor regression. In contrast, MRT-2359 had limited or no activity in low N-Myc NSCLC models, further corroborating the selective vulnerability of Myc-driven tumors to GSPT1 degradation. Together these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven solid tumors addicted to the protein translation machinery and warrant rapid evaluation towards the clinic. Citation Format: Gerald Gavory, Mahmoud Ghandi, Anne-Cecile d’Alessandro, Debora Bonenfant, Agustin Chicas, Frederic Delobel, Brad Demarco, Alexander Flohr, Christopher King, Anne-Laure Laine, Vittoria Massafra, Rajiv Narayan, Arnaud Osmont, Giorgio Ottaviani, Dave Peck, Sarah Pessa, Nooreen Rubin, Thomas Ryckmans, Martin Schillo, Ambika Singh, Simone Tortoioli, Dominico Vigil, Vladislav Zarayskiy, John Castle, Filip Janku, Owen Wallace, Silvia Buonamici, Bernhard Fasching. Identification of MRT-2359 a potent, selective and orally bioavailable GSPT1-directed molecular glue degrader (MGD) for the treatment of cancers with Myc-induced translational addiction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3929.

With the advances in sequencing technologies, a huge amount of biological data is extracted nowadays. Analyzing this amount of data is beyond the ability of human beings, creating a splendid opportunity for machine learning methods to grow. The methods, however, are practical only when the sequences are converted into feature vectors. Many tools target this task including iLearnPlus, a Python-based tool which supports a rich set of features. In this paper, we propose a holistic tool that extracts features from biological sequences (i.e. DNA, RNA and Protein). These features are the inputs to machine learning models that predict properties, structures or functions of the input sequences. Our tool not only supports all features in iLearnPlus but also 30 additional features which exist in the literature. Moreover, our tool is based on R language which makes an alternative for bioinformaticians to transform sequences into feature vectors. We have compared the conversion time of our tool with that of iLearnPlus: we transform the sequences much faster. We convert small nucleotides by a median of 2.8X faster, while we outperform iLearnPlus by a median of 6.3X for large sequences. Finally, in amino acids, our tool achieves a median speedup of 23.9X.

AbstractBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation.Significance:Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.This article is highlighted in the In This Issue feature, p. 275

In cancer, telomere maintenance is critical for the development of replicative immortality. Using genome sequences from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer Project, we calculated telomere content across 1299 cancer cell lines. We find that telomerase reverse transcriptase (TERT) expression correlates with telomere content in lung, central nervous system, and leukemia cell lines. Using CRISPR/Cas9 screening data, we show that lower telomeric content is associated with dependency of CST telomere maintenance genes. Increased dependencies of shelterin members are associated with wild-type TP53 status. Investigating the epigenetic regulation of TERT, we find widespread allele-specific expression in promoter-wildtype contexts. TERT promoter-mutant cell lines exhibit hypomethylation at PRC2-repressed regions, suggesting a cooperative global epigenetic state in the reactivation of telomerase. By incorporating telomere content with genomic features across comprehensively characterized cell lines, we provide further insights into the role of telomere regulation in cancer immortality.

Abstract The Myc family of transcription factors is a well-established driver of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy directly targeting the Myc family members has been developed to date. Abnormal activation of Myc results in uncontrolled cell growth that is associated with high translational output and ramp up of the protein translational machinery. This creates a dependency to protein translation and in turn represents a potential therapeutic vulnerability for Myc-driven tumors. Based on these considerations, we hypothesized that targeting the translational termination factor GSPT1, a key player of protein synthesis, may constitute a vulnerability for Myc-driven tumors. Using our proprietary Quantitative and Engineered Elimination of Neosubstrates (QuEENTM) platform we characterized and explored the known G-loop degron in GSPT1 that renders it amenable to cereblon-induced degradation by molecular glue degraders (MGDs). We rationally designed and subsequently screened a proprietary library of cereblon-binding small molecules, including GSPT1-directed MGDs, in human mammary epithelial cells (HMECs) expressing doxycycline-inducible c-Myc. Doxycycline treatment led to sustained c-Myc expression and as a consequence to the induction of key biomarkers of enhanced protein translation, such as phospho 4EBP1 (p4EBP1). We identified MRT-048 as a potent and highly selective GSPT1 degrader and demonstrated its ability to induce cell death in Myc-driven HMEC cells whilst sparing control cells (EC50 0.64 μM vs 30 μM respectively). This confirmed the selective vulnerability of Myc-driven cell growth to GSPT1 degradation. In follow-up studies, we confirmed the correlation between p4EBP1 as biomarker of Myc-activation and sensitivity to MRT-048 in a large panel of breast cancer cell lines. Moreover, MRT-048 treatment of animals xenografted with breast cancer cells induced tumor regression and was associated with complete GSPT1 degradation. Mechanistically, we observed that GSPT1 degradation induced by MRT-048 led to inhibition of genes regulated by Myc and ribosomal stalling at stop codons of several mRNAs. Additionally, polysome profiling of cancer cells treated with MRT-048 was associated with a global reduction of the intensities of the polysome peaks and concomitant increase in the monosome peaks as previously observed in GSPT1 knockdown experiments, suggesting that GSPT1 degradation by our MGD molecules affects both the termination and initiation stages of protein translation. We believe these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven tumors dependent on protein translation machinery. Citation Format: Gerald Gavory, Bernhard Fasching, Debora Bonenfant, Amine Sadok, Ambika Singh, Martin Schillo, Vittoria Massafra, Anne-Cecile d’Alessandro, John Castle, Mahmoud Ghandi, Agustin Chicas, Frederic Delobel, Alexander Flohr, Giorgio Ottaviani, Thomas Ryckmans, Anne-Laure Laine, Oliv Eidam, Hannah Wang, Ilona Bernett, Laura Chan, Chiara Gorrini, Theo Roumiliotis, Jyoti Choudhary, Yann-Vai LeBihan, Marc Cabry, Mark Stubbs, Rosemary Burke, Rob Van Montfort, John Caldwell, Rajesh Chopra, Ian Collins, Silvia Buonamici. Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA004.

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate or the expression of a marker gene. Information-rich assays, such as gene-expression profiling, are generally not amenable to efficient profiling of a given perturbation across multiple cellular contexts. Here, we developed 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, and combine it with Cell Hashing to further multiplex additional experimental conditions, such as multiple 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 can be used to predict long-term cell viability from short-term transcriptional responses to treatment.