Introduction: Patients with relapsed or refractory (R/R) DLBCL have a poor prognosis, even if treated with salvage chemotherapy and autologous stem cell transplantation (ASCT). CAR T therapy provides long-term remission in only 40% of patients, and some cannot receive CAR T therapy due to fitness, geographic, or financial limitations. Epcoritamab is a subcutaneous CD3×CD20 bispecific antibody approved for adults with R/R large B-cell lymphomas and follicular lymphoma after ≥2 lines of systemic therapy. Combining epcoritamab with lenalidomide provides a chemotherapy-free regimen that may enhance T-cell proliferation and augment natural killer cell activity, thereby increasing antitumor cytotoxicity. EPCORE NHL-5 (NCT05283720) is an ongoing, phase 1b/2, open-label, multi-arm, dose-escalation/expansion trial of epcoritamab in combination with antineoplastic agents for the treatment of non-Hodgkin lymphoma. Preliminary results from arm 1 of EPCORE NHL-5 demonstrated antitumor activity and tolerable safety in patients with R/R DLBCL treated with epcoritamab plus lenalidomide (Avivi Mazza, et al. Blood. 2023;142[Suppl1]:438). Here, we report updated results. Methods: Eligible patients were ≥18 years old with CD20+ R/R DLBCL, had an Eastern Cooperative Oncology Group performance status of 0-2, were previously treated with at least 1 systemic therapy containing an anti-CD20 antibody, and were ineligible for or failed ASCT. Patients received epcoritamab (cycles 1-3, once weekly; cycles 4-12, once every 4 weeks) and oral lenalidomide (25 mg/day on days 1-21) for a total of twelve 28-day cycles. Epcoritamab was administered with two step-up doses in cycle 1: day 1, 0.16 mg; day 8, 0.8 mg; followed by 48 mg full dose from day 15 onward. Corticosteroid prophylaxis for cytokine release syndrome (CRS) was required during cycle 1. Key endpoints included dose-limiting toxicities (DLTs), investigator-assessed response (overall response rate [ORR] and complete response [CR] rate), duration of response (DOR), time to response, and safety. Results: At the cutoff date of January 18, 2024, 40 patients received epcoritamab plus lenalidomide (60% male; median age 71.5 years, range, 26-85). Median (range) follow-up time was 11.5 (0.3-16.8) months. Among response-evaluable patients (n=37), ORR was 67.6%, with 51.4% of patients achieving a CR. Median DOR and CR have not been reached. Consistently high CR rates were observed across subgroups, including second-line patients (56.3%; n=16), patients who received prior CAR T (50%; n=10), and by molecular origin classification (GCB, 46.7%, n=15; ABC/non-GCB/unclassified, 46.7%, n=15). The safety profile was similar to that previously presented (Avivi Mazza, et al. Blood. 2023;142[Suppl1]:438). The most common grade 3-4 treatment-emergent adverse events (TEAEs) were neutropenia (60%), anemia (20%), thrombocytopenia (17.5%), and CRS (10%). Febrile neutropenia occurred in 2 patients (5%). Two patients (5%) experienced DLTs: CRS (Gr 3; n=1) and increased aspartate aminotransferase (Gr 4; n=1) during dose expansion. Overall, 65% of patients experienced CRS, mostly low grade: Gr 1: 14 (35%), Gr 2: 8 (20%), Gr 3: 4 (10%). Median onset of CRS was 16 days from the first dose (1 day after first full dose of epcoritamab) and all events resolved with a median time to resolution of 2 days. Immune effector cell-associated neurotoxicity syndrome occurred in 1 patient (2.5%; Gr 3), which resolved in 3 days. CRS rates and peak IL-6 levels were lower in patients receiving dexamethasone vs prednisone for CRS prophylaxis. There was 1 grade 5 TEAE considered related to epcoritamab of COVID-19 pneumonia. TEAEs leading to discontinuation of epcoritamab occurred in 4 (10%) patients (thrombocytopenia, n=2; COVID-19 pneumonia, n=1; pneumonia pneumococcal, n=1). Conclusion: With longer follow-up, the combination of fixed-duration epcoritamab with lenalidomide continues to demonstrate deep and durable responses with a manageable safety profile for R/R DLBCL. CRS was predictable and mostly low grade; of note, most patients received prednisone vs the recommended dexamethasone for CRS prophylaxis. This chemotherapy-free combination may provide an alternative option to platinum-based or other standard of care therapies and thus supports the ongoing phase 3 trial of this combination for the treatment of R/R DLBCL (NCT06508658).

AbstractPurpose: Seizure-related homolog protein 6 (SEZ6) is a novel target expressed in small cell lung cancer (SCLC). ABBV-011, a SEZ6-targeted antibody conjugated to calicheamicin, was evaluated in a phase I study (NCT03639194) in patients with relapsed/refractory SCLC. We report initial outcomes of ABBV-011 monotherapy. Patients and Methods: ABBV-011 was administered intravenously once every 3 weeks during dose escalation (0.3–2 mg/kg) and expansion. Patients with SEZ6-positive tumors (≥25% of tumor cells with ≥1+ staining intensity by IHC) were preselected for expansion. Safety, tolerability, antitumor activity, and pharmacokinetics were evaluated. Results: As of August 2022, 99 patients received ABBV-011 monotherapy [dose escalation, n = 36; Japanese dose evaluation, n = 3; dose expansion, n = 60 (1 mg/kg, n = 40)]; the median age was 63 years (range, 41–79 years). Also, 32%, 41%, and 26% of patients received 1, 2, and ≥3 prior therapies, respectively. The maximum tolerated dose was not reached through 2.0 mg/kg. The most common treatment-emergent adverse events were fatigue (50%), nausea (42%), and thrombocytopenia (41%). The most common hepatic treatment-emergent adverse events were increased aspartate aminotransferase (22%), increased γ-glutamyltransferase (21%), and hyperbilirubinemia (17%); two patients experienced veno-occlusive liver disease. The objective response rate was 19% (19/98). In the 1-mg/kg dose-expansion cohort (n = 40), the objective response rate was 25%; the median response duration was 4.2 months (95% confidence interval, 2.6–6.7); and the median progression-free survival was 3.5 months (95% confidence interval, 1.5–4.2). Conclusions: ABBV-011 1.0 mg/kg every 3 weeks monotherapy was well tolerated and demonstrated encouraging antitumor activity in heavily pretreated patients with relapsed/refractory SCLC. SEZ6 is a promising novel SCLC target and warrants further investigation.

Results from the single-target sgRNA screen in A549-Cas9 cells. The columns are as follows. Symbol : HUGO gene symbol ; EnsemblID : EnsEMBL identifier ; GeneID : Entrez Gene ID ; GeneInfo : Name of protein encoded by the gene ; Localization : GO cellular localization terms ; Process : GO cellular process terms ; Function : GO molecular function terms ; #Element: Number of identified sgRNAs in sequencing ; Phenotype (pZ) : median log2-fold enrichment of sgRNAS targeting this gene over the course of the experiment ; Tscore : GIT score ; MWU p-Val : p-value of gene depletion or enrichment by Mann-Whitney U test relative to Safe-sgRNAS ; MWU Adjusted Pval : FDR as determined by the Benjamini-Hochberg method.

Results from the first pairwise sgRNA screen in A549-Cas9 and H23-Cas9 cells. Columns are as in the previous table, except that they refer to Gene A or Gene B as indicated, where the symbol for GeneA comes alphabetically before the symbol of Gene B. Test statistics reflect comparisons with inferred expected phenotypes from Gene A/safe or Gene B/safe sgRNA pairs (see Methods).

Primers for quickchange mutagenesis of the indicated bait proteins used in AP/MS experiments.

Unbiased shRNA library screening and global transcriptome analyses reveal mechanisms that modulate dasatinib sensitivity and suggest therapeutic strategies to improve outcome of patients with acute lymphoblastic leukemia.

This table lists genes whose protein products were identified by AP/MS experiments in HEK293 cells or A549 cells on the indicated sheets. The columns are as follows. Bait: bait protein (gene-wise); Prey, prey protein (gene-wise); Background Values : NSAFs for other experiments, used to infer the background distributions as described above; Qualifier: mutation state of bait protein; FDR: false discovery rate; NSAF : the log10 transform of the Normalized Spectral Abundance Factor (see Methods), with greater numbers indicating stronger signals.

Background: The prognosis for patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) is poor, even if treated with salvage chemotherapy and autologous stem cell transplantation. CAR T cell therapy provides long-term remission in only 40% of patients. Epcoritamab, a subcutaneous CD3xCD20 bispecific antibody developed using the DuoBody ® platform, received accelerated FDA approval for adults with R/R DLBCL not otherwise specified, including DLBCL arising from indolent lymphoma, and high-grade B-cell lymphoma after ≥2 lines of systemic therapy. Single-agent epcoritamab has demonstrated deep and durable responses (overall response rate, 63%; complete responses, 40%) and a manageable safety profile in patients with R/R large B-cell lymphoma in the EPCORE NHL-1 trial (Karimi et al, ASCO 2023, abstract 7525). Combining epcoritamab with antineoplastic agents that have different mechanisms of action may offer enhanced clinical benefit to patients. Therefore, EPCORE NHL-5 (NCT05283720) evaluated the safety, tolerability, and preliminary efficacy of epcoritamab, as well as defining its recommended dose when coadministered with other antineoplastic agents in patients with non-Hodgkin lymphoma. Here, we present results from arm 1 that evaluated epcoritamab combined with lenalidomide in patients with R/R DLBCL. Methods: EPCORE NHL-5 is a phase 1b/2, nonrandomized, open-label, multicenter study. In arm 1, adults with CD20 + R/R DLBCL (ECOG 0-2) received subcutaneous epcoritamab (weekly during cycle 1-3, every 4 weeks during cycle 4-12) and oral lenalidomide (once daily on days 1-21) for 12 cycles of 28 days. Patients had received at least 1 prior combined systemic therapy that contained an anti-CD20 monoclonal antibody. The primary endpoint was the identification of dose-limiting toxicities (DLTs). Key secondary endpoints included investigator-assessed best overall response by Lugano 2014 criteria and time to response. Safety endpoints included severity and incidence of adverse events (AEs) including AEs of special interest to epcoritamab (cytokine release syndrome [CRS], immune cell-associated neurotoxicity syndrome [ICANS], and clinical tumor lysis syndrome). Results: As of May 22, 2023, 26 patients (median age, 71 years; DLBCL, n=24 [92%]; follicular lymphoma grade [G] 3b, n=2 [8%]) had received epcoritamab plus lenalidomide. Median duration of epcoritamab exposure was 3.8 months (range, 0-7.5) and median duration of lenalidomide exposure was 4.0 months (range, 0.1-8.2); 15 patients (58%) remain on epcoritamab and lenalidomide. Median number of prior lines of therapy was 1 (range, 1-4), with 15 (58%) receiving 1 prior line of anticancer therapy. Six patients (23%) had received prior CAR T and 2 patients had undergone hematopoietic stem cell transplantation. One DLT (neutropenia) was observed. The most common G3-4 treatment-emergent AEs (TEAEs) were neutropenia (n=15; 58%), anemia (n=4; 15%), thrombocytopenia (n=4; 15%), and febrile neutropenia (n=3; 12%). Primary granulocyte colony-stimulating factor prophylaxis was not mandatory. TEAEs led to epcoritamab discontinuation in 1 patient (3.8%; thrombocytopenia). No patients experienced a TEAE leading to death that was considered related to epcoritamab. CRS was predominantly low grade (73% [19/26] any grade; 65% G1-2; 8% G3) and occurred mostly after the first full dose (C1D15). Preliminary biomarker analysis showed pharmacodynamic profiles consistent with the mechanism of action of epcoritamab. These include predictable cytokine peaks immediately after the first full dose (IFN-gamma, IL-2, and IL-6) with a rapid and sustained depletion of peripheral B cells. One patient experienced ICANS (G3), which resolved after 2 days. Among response-evaluable patients (n=24), the overall response rate was 75% (95% CI, 53.3, 90.2). Complete metabolic responses were seen in 14 patients (58%) and partial responses in 4 patients (17%), of which 3 were ongoing at time of data cutoff ( Table). Median time to first response was 1.8 months (range, 1.0-2.8). Follow-up is ongoing. Conclusions: Epcoritamab combined with lenalidomide showed promising antitumor activity with a tolerable safety profile in patients with R/R DLBCL.

This spreadsheet contains 6 tabs, with each tab corresponding to a cell line and GO namespace. For each indicated bait (columns), all hits detected with FDR{less than or equal to}0.05 were searched for enrichment for the indicated term (row) using a background set of all protein-coding genes for which at least one GO term existed by Fisher's Exact Test. The values of the table are the FDRs derived from p-values by the Benjamini-Hochberg method.

3002 Background: Small cell lung cancer (SCLC) has a dismal prognosis and new therapies are urgently needed. SEZ6 is a transmembrane protein expressed in SCLC tumors that may be used as a therapeutic target. ABBV-011 is an antibody-drug conjugate (ADC) targeting SEZ6 with a calicheamicin payload, which has shown antitumor activity in preclinical models of SCLC. Preliminary results from the monotherapy dose-escalation and -expansion cohorts of the first-in-human ABBV-011 study are presented. Methods: Phase 1, open-label, multicenter study (NCT03639194) of ABBV-011 alone or in combination with budigalimab, a programmed cell death 1 inhibitor. Primary objectives were to assess the safety and tolerability and to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose of ABBV-011. Adults (≥18 years) with relapsed/refractory SCLC (1–3 lines of prior therapy) were enrolled. Dose escalation was guided by Bayesian continual reassessment method. ABBV-011 was administered intravenously at doses from 0.3 to 2.0 mg/kg once every 3 weeks. Dose expansion was conducted in SEZ6-selected patients. Results: At data cutoff on August 22, 2022, 99 patients were treated with ABBV-011 monotherapy. Median age was 63 years (range, 41–79), 50% of patients were male, and 68% had received ≥2 prior therapies. ABBV-011 ADC pharmacokinetics were approximately dose-proportional with an elimination half-life of 4.6 days across the dose range of 0.3–2.0 mg/kg. In dose escalation (n=26), 1 patient had a dose-limiting toxicity of grade (G) 3 fatigue at 2.0 mg/kg. We report safety and efficacy results for 40 patients in the dose-expansion 1.0-mg/kg ABBV-011 cohort. Median duration of treatment was 12 weeks (range, 1.9–63.3). Treatment-emergent adverse events (TEAEs) occurred in 39 (98%) patients, the most frequent being fatigue (48%), nausea (45%), anorexia (38%), thrombocytopenia (38%), and vomiting (35%). G3 TEAEs occurred in 18 (45%) patients, the most frequent being fatigue, thrombocytopenia, and neutropenia (10% each); 1 G4 TEAE of dyspnea was reported. Seven patients died due to malignant neoplasm/disease progression (n=6) or respiratory distress (n=1); none were related to ABBV-011. Hepatotoxicity was observed, including G≥2 TEAEs of hyperbilirubinemia (18%), increased gamma-glutamyltransferase (8%), ascites (5%), veno-occlusive liver disease (3%), and portal hypertension (3%). Confirmed objective response rate was 25% (10 partial responses [PR]), with median duration of response of 4.2 months (95% CI: 2.6, 6.7). Clinical benefit rate (CBR) was 65% (10 PR and 16 stable disease) and CBR lasting >12 weeks was 43%. The median progression-free survival was 3.5 months. Conclusions: The MTD was not reached and ABBV-011 was well tolerated at 1.0 mg/kg with promising antitumor activity observed. Further evaluation of ABBV-011 is ongoing. Clinical trial information: NCT03639194 .

e21105 Background: Biomarker-targeted antibody-drug conjugates (ADCs) have shown promise in treating lung cancer. Telisotuzumab vedotin (Teliso-V; ABBV-399) is a first-in-class MET (also known as c-Met)-directed ADC comprising the monoclonal antibody telisotuzumab (ABT-700) conjugated to a cytotoxic microtubule inhibitor, monomethyl auristatin E (MMAE), via a cleavable dipeptide linker. In the phase 2 LUMINOSITY study (NCT03539536), Teliso-V demonstrated promising anticancer activity in previously treated patients with MET-overexpressing (OE), non-squamous epidermal growth factor receptor wild type ( EGFR WT) non-small cell lung cancer (NSCLC; 52.2% overall response rate in MET OE high group, 36.5% in all MET OE cohort [intermediate and high]) and an acceptable safety profile (Camidge et al. J Clin Oncol. 2022;40:16 suppl, 9016). These data provide proof of concept that biomarker-selected MMAE-based ADCs could be beneficial to patients with NSCLC. Cofetuzumab pelidotin (ABBV-647), an ADC with a similar composition to Teliso-V, contains an anti-protein tyrosine kinase 7 (PTK7) monoclonal antibody (hu6MO24) conjugated to a microtubule-inhibiting cytotoxin, Aur0101 auristatin, via a cleavable cysteine-reactive linker. PTK7 expression and oncogenic functions have been reported in several cancers. We sought to establish the co-prevalence of MET OE and PTK7 in NSCLC to understand if both these antigens targeted by ADCs are co-expressed or present in distinct tumors. Methods: Antigen prevalence was analyzed in tumor samples from patients at the City of Hope National Medical Center with non-squamous NSCLC and EGFR WT or unknown status. Immunohistochemistry (IHC) assays used were MET (SP44) Assay for MET OE (Roche Tissue Diagnostics; positive if ≥25% cells at 3+ intensity) and an AbbVie-developed assay for PTK7 expression (positive if ≥90% cells at ≥2+ intensity). Results: A total of 148 patients (median age 67 years, 52% stage ≥III at diagnosis, 68% ECOG 0–2) were screened (data shown in table). About 24% of the patients were MET IHC positive, whereas 11% were PTK7 IHC positive. Further, MET OE and PTK7 had complementary prevalence with only 3% of the patients co-expressing both antigens. Conclusions: MET OE and PTK7 expression by IHC were found to be complementary in patients with non-squamous EGFR WT/unknown NSCLC. About 32% of patients were positive for MET OE or PTK7 expression or both. Data represent patients from a single center and more pre-treatment tissue samples that were viable and available. These IHC findings suggest that MET OE and PTK7 are indeed complementary NSCLC biomarkers. [Table: see text]

The fungal pathogen Histoplasma capsulatum ( Hc ) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc . We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc , the ubiquitous fungal pathogen Candida albicans , and the causative agent of Valley Fever Coccidioides posadasii . We showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc -macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

TPS3142 Background: Protein tyrosine kinase 7 (PTK7) is a highly conserved receptor tyrosine kinase involved in the Wnt signaling pathway and is overexpressed in multiple cancer types, including non-small cell lung cancer (NSCLC). Cofetuzumab pelidotin (ABBV-647) is an anti-PTK7 antibody-drug conjugate comprising the hu6MO24 monoclonal antibody, a cleavable cysteine-reactive linker, and Aur0101 (an auristatin microtubule inhibitor). It has shown promising preclinical anti-tumor effects (Damelin et al. Sci Transl Med 2017;9[372]:eaag2611) and clinical activity with a manageable safety profile in a Phase 1 study in patients with advanced solid tumors, with promising anti-tumor activity noted in NSCLC (Sachdev et al. DOI: 10.1200/JCO.2018.36.15_suppl.5565). Methods: This open-label, single-arm, multicenter Phase 1b study (NCT04189614) will assess the anti-tumor activity and safety of cofetuzumab pelidotin in approximately 40 patients with PTK7-expressing, recurrent NSCLC. The primary objective is to assess the objective response rate of cofetuzumab pelidotin according to Response Evaluation Criteria in Solid Tumors version 1.1. Secondary objectives include the duration of response, progression-free survival, overall survival, and safety and tolerability. Pharmacokinetic and biomarker samples will also be collected throughout for analysis. Patients must be aged ≥18 years with an Eastern Cooperative Oncology Group performance status of 0–1 and have recurrent histologically confirmed NSCLC with PTK7-expressing tumor (using a validated immunohistochemistry assay). Patients must have progressed after treatment with a platinum-based chemotherapy doublet and an immune checkpoint inhibitor (for tumors without targetable genetic alterations), or a platinum-based chemotherapy doublet and targeted agent(s) (for tumors with targetable genetic alterations). Patients must also have received ≤2 prior lines of systemic therapy (≤3 prior lines for tumors treated with targeted agent[s] for genetic alterations), including no more than 1 line of systemic chemotherapy. Cofetuzumab pelidotin (2.8 mg/kg) is administered intravenously every 3 weeks until the patient experiences disease progression, intolerable toxicity, or other study treatment discontinuation criteria are met. The study commenced on February 13, 2020 and enrollment is ongoing. Clinical trial information: NCT04189614.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication 1 . Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D–CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer 2 , 3 . However, the mechanisms that regulate levels of cyclin D are incompletely understood 4 , 5 . Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

Abstract Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein–protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. Significance: We establish a deep network of protein–protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers. This article is highlighted in the In This Issue feature, p. 1775

Abstract Synthetic lethal-based approaches to targeting KRAS driven cancers have recently been receiving increasing attention. However, as KRAS activates multiple effector pathways, targeting single genes may not be sufficient to fully inhibit KRAS-driven oncogenesis. In addition, genome-wide screens can be limited by high noise and low signal. Furthermore, it is increasingly clear that standard screens based on 2D proliferation can miss critical phenotypes relevant to cancer biology. Therefore, we developed a multistage screening strategy that includes a) identification of KRAS-proximal protein-protein interaction (PPI) networks, b) targeted CRISPR-based screening for pairs of genes that together are synthetic lethal with KRAS, and c) validation in a 3D sphere system using both human and mouse NSCLC cells. We used affinity purification/mass spectrometry (AP/MS) to construct a detailed map of protein-protein interactions centered on KRAS. Based on this network, we designed a CRISPR/Cas9 library targeting pairwise combinations of 119 KRAS-interacting genes. We screened two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). This screen yielded a large number of gene pairs that synergistically impaired cell growth. We selected the 20 most promising targets for further screening in a panel of 5 KRAS-mutant and 4 KRAS wild-type NSCLC cell lines. From these screens we identified a KRAS-specific synergistic interaction between two genes—Rap1GDS1 and RhoA. Validation in 3D cultures in a panel of cell lines showed that combinatorial Rap1GDS1 and RhoA knockout selectively impairs sphere growth of KRAS-mutant NSCLC cells but has minimal effects on KRAS wild-type NSCLC cell lines or nontransformed lung cells. We demonstrate that this effect is primarily due to the induction of apoptosis. Rap1GDS1 has previously been reported to encode two different isoforms with distinct cellular functions. We observe that only one of the isoforms is involved in the mediation of the KRAS-dependent lethal effect. Current efforts are directed at elucidating the mechanistic basis for the isoform-specific combinatorial lethality between Rap1GDS1 and RhoA. Furthermore, we are actively exploring how to exploit this knowledge to define potential novel therapies for KRAS-driven cancer. Overall, we demonstrate that an integrated strategy combining proteomics, paired CRISPR screens and innovative 3D validation systems can identify novel synthetic lethal combinations. This approach may help identify novel synthetic lethal drug combinations for KRAS and other to-date undruggable oncogenes. This abstract is also being presented as Poster A40. Citation Format: Kaja Kostyrko, Marcus Kelly, Kyuho Han, Edwin Jeng, David Morgens, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Combinatorial knockout of Rap1GDS1 and RhoA leads to lethality in KRAS-driven non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR07.

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.

The RAS oncogenes are mutated in one third of human cancers, but therapies against Ras-driven cancers have been unsuccessful. Ras proteins themselves are not yet druggable, and the Ras pathway contains uncharacterized redundancies, feedback mechanisms, and tributaries that have stymied the development of other targeted therapies. Multiple-agent combination therapies hold some promise, but their development requires a more thorough understanding of Ras cell biology than we currently possess. We therefore pursued creation of a physical and genetic map of the Ras pathway in non-small-cell lung cancer (NSCLC). Using H, K-, and NRas and twelve other Ras pathway proteins as baits, we conducted tandem affinity purification experiments to create a high-confidence protein-protein interaction (PPI) network. We integrated our data with public PPI, genetic susceptibility, and patient data to assemble an interpretable interaction map encompassing 360 proteins and 1000 physical interactions among them. Guided by the topology and annotations of this PPI map, we constructed a library of 1000 sgRNAs covering 120 genes. These were screened for pairwise genetic interactions (GIs) in A549 and H23 NSCLC lines using a dual sgRNA vector system to discover over 250 synthetic lethal genetic interactions. Each suggests a strategy for combination therapy. The combined GI/PPI network also produced myriad mechanistic hypotheses. Pursuing these, we made new discoveries that demonstrate the power of the approach. First, we find that KRas binds the cell adhesion regulator Radil, and that Ras regulates cell morphology by modulating the Rap signaling pathway. Second, we identify the critical guanine nucleotide exchange factor and effector by which KRas up-regulates macropinocytosis. Third, we demonstrate that that the in vivo physical interactions between Ras, Raf, and RalGEF family proteins depend crucially on the specific paralogs involved. The equivalent in vitro interactions are not selective, demonstrating that many undiscovered factors direct the strong specificity observed in vivo. Distinct genetic interaction patterns between paralogs in these same families support the non-equivalence of their members. Fourth, we detect a new synthetic lethal genetic interaction between the GTPase chaperone Rap1GDS1 and the GTPase RhoA. The strength of interaction varies among NSCLC lines, but correlates with the KRas dependence of each. This and other interactions show that several other small GTPases work in concert with Ras signaling to regulate tumor progression. Together, these discoveries show that our multiomic approach furthers two critical goals: it produces testable hypotheses that point to new Ras cell biology and reveals combination susceptibilities for the development of new therapies against Ras-driven cancers. Citation Format: Marcus R. Kelly, Kyuho Han, Kaja Kostyrko, Edwin E. Jeng, Nancie Mooney, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. Proteomic and genetic interaction mapping of the Ras pathway reveals new effectors and vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 959.

Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS, RIN1/2) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 “safe harbor” control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs, identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing, and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation, and protein processing, including the RADIL, RGL, and RIN Kras effectors. Validation focused using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf, and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathways including cell adhesion (RADIL), growth signaling (RAF), and endocytosis/ macropinocytosis (RIN) are affected and that use of the Kras inhibitor ARS-853 shows much reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates. This abstract is also being presented as Poster A28. Citation Format: Marcus R. Kelly, Kyuho Han, Nancie Mooney, Edwin Jeng, Kaja Kostyrko, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR12.

Abstract Lung cancer is the number one cause of cancer-related deaths worldwide. The most prevalent type of lung cancer is Non-Small Cell Lung Cancer (NSCLC). A significant number of patients with NSCLC carry oncogenic KRAS mutations. However, the efforts to target KRAS directly have thus far proven unsuccessful and tumors harboring mutations in this gene remain the most difficult to treat, highlighting the need for alternative approaches. One promising strategy is to target KRAS-dependent cancers through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS oncogenesis. Therefore, we propose that targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic strategy. Furthermore, we hypothesize that a targeted approach focused on the protein-protein interaction network proximal to KRAS may be more effective than the current emphasis on genome-wide screens. To discover novel, combinatorial KRAS synthetic lethal genes, we used affinity purification/mass spectrometry (AP/MS), to systematically identify KRAS interacting proteins and construct a detailed map of protein-protein interactions centered on KRAS. Based on this network we designed a CRISPR/Cas9 library targeting pairwise combinations of KRAS-interacting genes. Using this library we simultaneously knocked-out pairs of 119 genes in two KRAS-driven non-small cell lung cancer (NSCLC) cell lines (A549 and H23). Knock-out of many gene pairs synergistically impaired growth of these cells, while the knock-out of each of the genes alone had no or little effect. We chose 20 most promising targets for further screening in vitro and in vivo in a panel of 9 KRAS-mutant and KRAS wild type Cas9-expressing NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth in A549 and H23 cells for individual validation in Cas9-expressing NSCLC cell lines and normal human bronchial epithelial cells (HBECs). We found that the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, significantly decreased growth of KRAS-dependent NSCLC cells, while having a limited effect on KRAS-independent cells or HBECs. Moreover the knock-out of either of these genes alone had no effect on growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are currently performing further validation in organoid cultures and in vivo. Additional validation and human relevance will be determined using patient-derived xenografts (PDX). Citation Format: Kaja Kostyrko, Marcus R. Kelly, Kyuho Han, Edwin E. Jeng, David W. Morgens, Michael C. Bassik, Peter K. Jackson, Alejandro Sweet-Cordero. Identification of novel combinatorial synthetic lethal vulnerabilities in KRAS-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4362.