Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFNγ-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFNγ pathway, allowing us to distinguish it as a novel mechanism of immune resistance.We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

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 singlecell 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 posttreatment 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 shortterm transcriptional responses to treatment.

No abstract

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.

Transcription activator-like effector proteins (TALEs) contain large numbers of repeats that bind double-stranded DNA, wrapping around DNA to form a continuous superhelix. Since unbound TALEs retain superhelical structure, it seems likely that DNA binding requires a significant structural distortion or partial unfolding. In this study, we use nearest-neighbor "Ising" analysis of consensus TALE (cTALE) repeat unfolding to quantify intrinsic folding free energies, coupling energies between repeats, and the free energy distribution of partly unfolded states, and to determine how those energies depend on the sequence that determines DNA-specificity (called the "RVD"). We find a moderate level of cooperativity for both the HD and NS RVD sequences (stabilizing interfaces combined with unstable repeats), as has been seen in other linear repeat proteins. Surprisingly, RVD sequence identity influences both the overall stability and the balance of intrinsic repeat stability and interfacial coupling energy. Using parameters from the Ising analysis, we have analyzed the distribution of partly folded states as a function of cTALE length and RVD sequence. We find partly unfolded states where one or more repeats are unfolded to be energetically accessible. Mixing repeats with different RVD sequences increases the population of partially folded states. Local folding free energies plateau for central repeats, suggesting that TALEs access partially folded states where a single internal repeat is unfolded while adjacent repeats remain folded. This breakage should allow TALEs to access superhelically-broken states, and may facilitate DNA binding.