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Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell–specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune–metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti–PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms.Significance:BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms.This article is highlighted in the In This Issue feature, p. 517

The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, antiproliferation, and proapoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ-resistance mechanisms, we performed IFNγ-sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.

Machine learning evaluation of immune-metabolic targets in predicting patient response to anti-PD1 and anti-CTLA4 combination therapy.

IFN-γ sensitivity of cancer cells (n = 43) from CCLE (sheet1); Gene sets enrichment for highly expressed gene sets in IFN-γ resistant/sensitive cell lines using CCLE transcriptomic dataset (sheet2-3).

The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, antiproliferation, and proapoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ-resistance mechanisms, we performed IFNγ-sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell–specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune–metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti–PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms.Significance:BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms.This article is highlighted in the In This Issue feature, p. 517

Supplementary Figures S1-S8

We detailed a comparison of BipotentR with competing and alternative approaches in Supplementary notes 1, 2, 6, and 7. Supplementary Note 3-5 describes the in vitro findings of ESRRA, the potential clinical relevance of ESRRA across cancer-types, and the association of tumor activity of ESRRA with immune infiltration.

RNA-seq and ATAC-seq of TRAF3-normal and -deficient B16F10 cells treated with vehicle control or IFNγ

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell–dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy.Significance:MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell–based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials.This article is highlighted in the In This Issue feature, p. 1307

Supplementary Figures S1-S8

Sup.Fig.9: The effect of Man2a1 inhibition on the immune cell infiltration.

Sup.Table.S3: Gene sets enriched in Man2a1-null B16F10 tumors vs control tumors treated with anti-PD-L1. Pre-ranked gene set enrichment analysis (GSEA) was used to perform the analysis.

Supplementary Figure Legend

Purpose:Immune checkpoint blockade has shown remarkable efficacy, but in only a minority of patients with cancer, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in antitumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes.Experimental Design:We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We further validated our findings using in vitro coculture and in vivo syngeneic tumor growth assays.Results:We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti–PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T-cell–mediated killing. Man2a1 knockout enhanced response to anti–PD-L1 treatment and facilitated higher cytotoxic T-cell infiltration in tumors under anti–PD-L1 treatment. Furthermore, a pharmacologic inhibitor of MAN2A1, swainsonine, synergized with anti–PD-L1 in syngeneic melanoma and lung cancer models, whereas each treatment alone had little effect.Conclusions:Man2a1 loss renders cancer cells more susceptible to T-cell–mediated killing. Swainsonine synergizes with anti–PD-L1 in suppressing tumor growth. In light of the limited efficacy of anti–PD-L1 and failed phase II clinical trial on swainsonine, our study reveals a potential therapy combining the two to overcome tumor immune evasion.See related commentary by Bhat and Kabelitz, p. 5778

Background: Platinum agents induce DNA crosslinking and cause accumulation of genotoxic stress, which leads to immune activation via IFN-γ signaling, making the combination with nivolumab (PD-1 antibody) an attractive strategy to enhance the benefit of either agent alone in metastatic triple-negative breast cancer (mTNBC). Methods: In this phase II open-label, investigator-initiated, multicenter trial, patients with unresectable locally advanced or mTNBC treated with 0-1 prior lines of chemotherapy in the metastatic setting were randomized 1:1 to carboplatin (AUC 6) with or without nivolumab (360 mg) IV every 3 weeks. Stratification factors included: germline BRCA (gBRCA) status, prior neo/adjuvant platinum, and number of prior lines of metastatic therapy. After approval of PD-L1 inhibition for mTNBC, the study was amended to include first-line mTNBC only and PD-L1 status was added as a stratification factor. Patients randomized to carboplatin alone were allowed to crossover at progression to receive nivolumab (+ nab-paclitaxel post-amendment). The primary objective was to compare progression-free survival (PFS) per RECIST 1.1 criteria of carboplatin with or without nivolumab in first-line mTNBC in the intent-to-treat (ITT) population. Key secondary objectives were objective response rate (ORR), overall survival (OS), clinical benefit rate, and duration and time to objective response. PD-L1 status was confirmed centrally using the SP142 Ventana assay (positive, ≥1% IC). Paired research biopsies at baseline, on-treatment and at progression were performed, if safely accessible. The trial closed to accrual prior to reaching target accrual due to approval of PD-1 inhibition in combination with platinum-based chemotherapy for PD-L1+ mTNBC. Results: Between 1/30/2018 and 12/9/2020, 78 patients enrolled. Three patients did not receive protocol treatment, and the safety analysis was conducted among the 75 that received any treatment; 37 received carboplatin + nivolumab (Arm A), 38 received carboplatin alone (Arm B). Median age was 59.1 yrs (range: 25.4-75.8). Four patients (5.3%) had a known gBRCA1/2 mutation. Sixty-two (82.7%) patients received 0 prior lines (ITT population) and 13 (17.3%) 1 prior line of metastatic therapy. Sixty-seven patients (89.3%) experienced any grade ≥2 treatment-related adverse event (AE). The most frequent AE were platelet count decrease (n=40; 53.3%), anemia (n=36; 48.0%), neutrophil count decrease (n=33; 44.0%) and fatigue (n=24; 32.0%). Grade 3/4 AE were observed in 46 (61.3%) patients, and there was one grade 5 AE (COVID19 pneumonia). Any grade ≥2 immune-related AE (irAE) were observed in 25 of the 37 (67.6%) patients treated with carboplatin + nivolumab. Grade 3/4 irAE were observed in 11 (29.7%) patients. In the ITT population (32 on Arm A; 30 on Arm B), median PFS was 4.2 months with carboplatin + nivolumab, and 5.5 months with carboplatin (stratified HR 0.98, 95% CI [0.51 - 1.88]; p=0.95). ORR was 25% vs. 23.3%, respectively. At a median follow-up of 23.5 months, median OS was 17.5 months vs. 10.7 months (stratified HR 0.63, 95% CI [0.32 - 1.24]; p=0.18). In patients with PD-L1+ mTNBC (13 on Arm A; 11 on Arm B), median PFS was 8.3 months and 4.7 months, respectively (stratified HR 0.63, 95% CI [0.21 - 1.89]; p=0.41). ORR was 23.1% vs. 27.3%, respectively. Median OS was 17.5 months vs. 9.6 months (stratified HR 0.59, 95% CI [0.20 - 1.75]; p=0.34). Conclusions: Addition of nivolumab to carboplatin in patients with previously untreated mTNBC, unselected by PD-L1 status, did not significantly improve PFS. A trend toward improved PFS and OS was observed in patients with PD-L1+ mTNBC. Tissue, blood and intestinal microbiome biomarker analyses are planned; bulk tumor and single-cell sequencing, and TCR sequencing in peripheral blood are ongoing. Clinical trial information: NCT03414684. Citation Format: Ana C Garrido-Castro, Noah Graham, Kevin Bi, Jihye Park, Jingxin Fu, Tanya Keenan, Edward Thomas Richardson, Ricardo Pastorello, Paulina Lange, Victoria Attaya, Robert Wesolowski, Natalie Sinclair, Zarah Lucas, Steve Lo, Nadine Tung, Meredith Faggen, Peter A Kaufman, Caroline C Block, Fred Briccetti, Madhavi Toke, Wendy Chen, Kai Wucherpfennig, Sascha Marx, Ye Tian, Judith Agudo, Jennifer L Guerriero, Stuart Schnitt, Nancy U Lin, Eric P Winer, Elizabeth A Mittendorf, Nabihah Tayob, Eliezer Van Allen, Sara M Tolaney. A randomized phase II trial of carboplatin with or without nivolumab in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-18.

PurposePredictive biomarkers of immune checkpoint inhibitors (ICIs) efficacy are currently lacking for non-small cell lung cancer (NSCLC). Here, we describe the results from the Anti–PD-1 Response Prediction DREAM Challenge, a crowdsourced initiative that enabled the assessment of predictive models by using data from two randomized controlled clinical trials (RCTs) of ICIs in first-line metastatic NSCLC.MethodsParticipants developed and trained models using public resources. These were evaluated with data from the CheckMate 026 trial (NCT02041533), according to the model-to-data paradigm to maintain patient confidentiality. The generalizability of the models with the best predictive performance was assessed using data from the CheckMate 227 trial (NCT02477826). Both trials were phase III RCTs with a chemotherapy control arm, which supported the differentiation between predictive and prognostic models. Isolated model containers were evaluated using a bespoke strategy that considered the challenges of handling transcriptome data from clinical trials.ResultsA total of 59 teams participated, with 417 models submitted. Multiple predictive models, as opposed to a prognostic model, were generated for predicting overall survival, progression-free survival, and progressive disease status with ICIs. Variables within the models submitted by participants included tumor mutational burden (TMB), programmed death ligand 1 (PD-L1) expression, and gene-expression–based signatures. The bestperforming models showed improved predictive power over reference variables, including TMB or PD-L1.ConclusionThis DREAM Challenge is the first successful attempt to use protected phase III clinical data for a crowdsourced effort towards generating predictive models for ICIs clinical outcomes and could serve as a blueprint for similar efforts in other tumor types and disease states, setting a benchmark for future studies aiming to identify biomarkers predictive of ICIs efficacy.Context summaryKey objectiveNot all patients with non-small cell lung cancer (NSCLC) eligible for immune checkpoint inhibitor (ICIs) respond to treatment, but accurate predictive biomarkers of ICIs clinical outcomes are currently lacking. This crowdsourced initiative enabled the robust assessment of predictive models using data from two randomized clinical trials of first-line ICI in metastatic NSCLC.Knowledge generatedModels submitted indicate that a combination of programmed death ligand 1 (PD-L1), tumor mutational burden (TMB), and immune gene signatures might be able to identify patients more likely to respond to ICIs. TMB and PD-L1 seemed important to predict progression-free survival and overall survival. Mechanisms including apoptosis, T-cell crosstalk, and adaptive immune resistance appeared essential to predict response.Relevance

Annonaceous acetogenins (ACGs) have attracted much attention because of excellent antitumor activity. However, the lack of selectivity and the accompanying serious toxicity have eventually prevented ACGs from entering clinical application. To decrease the side effects of ACGs, the cytotoxicity of ACGs on 10 types of tumor cell lines was investigated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test to identify one that was very sensitive to ACGs. Meanwhile, ACGs nanoparticles (ACGs-NPs) were prepared using poloxamer 188 (P188) as an excipient so as to solve the problem of poor solubility and the in vivo delivery of ACGs. ACG-NPs were 163.9±2.5 nm in diameter, negatively charged, and spherical with a high drug loading content (DLC) of 44.9±1.2%. MTS assays demonstrated that ACGs had strong cytotoxicity against JEG-3, HeLa, SiHa, MCF-7, A375, A2058, A875, U-118MG, LN- 229, and A431 cells, among which JEG-3 cell line was extremely sensitive to ACGs with a 50% inhibitory concentration (IC50) value of 0.26 ng/mL, a very encouraging discovery. ACGs-NPs demonstrated very good dose-dependent antitumor efficacy in a broad range of 45?1200 μg/kg on JEG-3 tumor-bearing mice. At a very low dose (1200 μg/kg), ACGs-NPs achieved a high tumor inhibition rate (TIR) of 77.6% through oral administration, displaying a significant advantage over paclitaxel (PTX) injections that are currently used as first-line anti-choriocarcinoma drugs. In the acute toxicity study, the half lethal dose (LD50) of ACGs-NPs was 135.5 mg/kg, which was over 100 times as of the effective antitumor dose, indicating good safety of ACGs-NPs. ACGs-NPs show promise as a new type of and potent anti-choriocarcinoma drug in the future.

Background: The use of chemotherapeutic drugs is restricted in the tumor-therapy because of the severely toxic and side effects among most important factors. The active herbal extracts are always used as a high dose while in the tumortherapy to achieve good anti-tumor effects. Hydrous icaritin has a high activity while there are few existing dosage forms as a result of low solubility in water and poor bioavailability. Results: The prepared hydrous icaritin nanorods (DP-HICT NRs) using mPEG2000-DSPE as a stabilizer, presented a narrow distribution of particle size with of 217 nm and a properly high drug-loading content of approximately 65.3±1.5%. A low dose of hydrous icaritin nano-formulation shows remarkable efficacy in cancer therapy (tumor inhibition rate: 61.36±10.80%) compared with the same dose of Paclitaxel injection (tumor inhibition rate: 66.80±4.43%), which approved as medicaments. Not only that, DP-HICT NRs can escape the clearance of the immune system and enhance targeting ability to the tumor site with only one excipient and such a low dose. Conclusions: This kind of nanoparticles contain a low dose of HICT used mPEG2000-DSPE as a stabilizer, while can achieve good tumor targeting as some active targeting agents and an anti-tumor effect as the PTX injection. There are broad prospects in drug safety, anti-tumor efficacy and even prognosis.

Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Translational oncology studies increasingly emphasize deriving hypotheses from single-cell datasets by evaluating groups of patients (e.g., treated vs. untreated, responders vs. non-responders, early vs. late-stage tumors) for differences in gene signatures/pathways to guide subsequent investigations. However, when evaluating these questions using tumor single-cell data, current statistical methods for this task are limited in two major aspects: (i) They are not representative of the hierarchical nature of tumor single-cell datasets (where cells are more similar within the patient than between patients); and (ii) They often do not contextualize statistical significance (p-values) in the context of variability that is expected biologically between patients. This can lead to molecular hypotheses that are skewed by patient-specific biology with high false positive rates. To address these challenges, we developed a nonparametric statistical method, BEANIE (group Biology EstimAtioN in sIngle cEll), for estimating group biology in single-cell transcriptomic datasets. BEANIE uses monte carlo simulations to estimate the p-value distribution for the gene signature of interest and normalizes with respect to a background distribution of the expected patient-to-patient variability. The method then performs leave-one-out cross-validation to infer robustness of the gene signatures to patient exclusion. We applied our method to public cancer single-cell datasets to evaluate candidate differences in tumor cell populations between known groups. When comparing tumor cell programs derived from treatment-naive vs. chemotherapy-treated Triple-Negative Breast Cancer (TNBC) patients, BEANIE identified upregulation of hallmark gene signatures for fatty acid metabolism and hypoxia to be statistically significant and robust in the treatment-naive group, indicative of response to chemotherapy treatment. We also compared BEANIE to conventional methods like Mann-Whitney U (MWU) test followed by Benjamini-Hochberg (BH) correction and Generalized Linear Models (GLM). MWU test and GLM also identified these signatures to be upregulated, but they additionally identified other immune cell gene signatures like T-cell markers, B-cell markers and NK cell markers, to also be upregulated, which was not expected since we were comparing between tumor cells. Similarly, BEANIE identified candidate tumor cell intrinsic programs relevant to hypotheses in lung cancer patients (early vs. late-stage) and melanoma patients (Immune Checkpoint Blockade (ICB)-naive vs. ICB-treated). Therefore, BEANIE greatly increased the specificity of analysis, and reduced false positive rates. This method is publicly available and may overcome existing methodological limitations to guide tumor-intrinsic hypothesis generation in rapidly growing clinical cancer single-cell cohorts. Citation Format: Shreya Johri, Kevin Bi, Breanna M. Titchen, Jingxin Fu, Jake Conway, Jett Crowdis, Natalie I. Volkes, Zenghua Fan, Lawrence Fong, Jihye Park, David Liu, Meng Xiao He, Eliezer M. Van Allen. Dissecting tumor cell programs through group biology estimation in clinical single-cell transcriptomics [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 1120.

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but many patients with impaired MHC-I expression remain refractory. Here, we combined FACS-based genome-wide CRISPR screens with a data-mining approach to identify drugs that can upregulate MHC-I without inducing PD-L1. CRISPR screening identified TRAF3, a suppressor of the NFκB pathway, as a negative regulator of MHC-I but not PD-L1. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell–dependent killing, and adds to ICB efficacy. Our findings provide preclinical rationale for treating tumors expressing low MHC-I expression with SMAC mimetics to enhance sensitivity to immunotherapy. The approach used in this study can be generalized to identify other drugs that enhance immunotherapy efficacy. Significance: MHC-I loss or downregulation in cancer cells is a major mechanism of resistance to T cell–based immunotherapies. Our study reveals that birinapant may be used for patients with low baseline MHC-I to enhance ICB response. This represents promising immunotherapy opportunities given the biosafety profile of birinapant from multiple clinical trials. This article is highlighted in the In This Issue feature, p. 1307

Objective: To retrospectively analyze the clinical utility of quantitative nursing measures of 10 cases of lower cranial nerves injury after cerebellopontine angle tumors surgery to provide the experience for improving the recovery rate and living quality of these patients. Methods: The clinical data of 10 cases of lower cranial nerves injury after cerebellopontine angle tumors surgery was analyzed. For problems such as dysphagia and dyspnea of these patients, the nursing care focused on strict monitoring, timely oxygen inhalation nursing, posture nursing, ventilator nursing, swallowing function training, etc. Results: After received quantitative care, 10 patients with lower cranial nerves injury after cerebellopontine angle tumors surgery were recovered well, and their symptoms such as dysphagia and dyspnea were gradually improved and safely discharged. Conclusion: Lower cranial nerves injury is one of the serious complications after removal of cerebellopontine angle tumors, which impacts the life and health of patients. Caregivers should accurately understand and analyze the symptoms, and quantitative and targeted nursing measures for posterior cranial nerves injury are helpful in the postoperative rehabilitation of patients and improve their living quality.

Objective: To investigate the therapeutic effect of Shenmai Injection on postoperative cerebral vasospasm in patients with ruptured aneurysms. Methods: Seventy patients undergoing craniotomy for ruptured aneurysms in our hospital were selected as study subjects and randomly divided into control (n = 33) and research (n = 37) groups, they were treated with nimodipine and nimodipine combined with Shenmai injection after operation. The blood flow velocity in the middle cerebral artery (MCA) before and at 1, 3, 7, 11 and 14 days after surgery and the incidence of cerebral vasospasm during these days were compared, and the GCS scores at 14 days postoperatively and GOS scores at 6 months postoperatively were compared between the two groups. Results: There were no statistically significant differences in the occurrence of cerebral vasospasm, GCS or GOS scores between the two groups (P > 0.05), but the period of postoperative cerebral vasospasm in the study group was significantly shorter than that in the control group. Conclusion: Shenmai injection has the effect of shortening the cycle of occurrence of cerebral vasospasm after the operation of ruptured aneurysms, promoting patients to recover as early as possible and reducing their physical and mental burden.

Rationale: The three-dimensional (3D) computer-made titanium mesh is widely used in the skull repair for those patients receiving decompression of the bone flap. It can restore normal anatomy to a greater extent and make a better appearance. Case Presentation: We reported two cases of patients admitted to our hospital who have experienced high scalp tension after skull repair. At first, these two patients underwent decompression of the bone flap, and the physical examination results showed a defect of skull. No neurological symptoms and signs were found. The 3D computed tomography (CT) reconstruction of skull was performed, and then the skull repair with 3D titanium mesh was conducted. But because of high scalp tension, they underwent a second operation, during which we re-trimmed and reduce the arc of the titanium mesh. The scalp incision of both patients healed well and no titanium mesh was exposed. Both patients have a good prognosis. Lessons: We highlight that the high tension of scalp due to overstretching after 3D titanium mesh repair for skull defect should be paid much attention to. Trimming and reducing the arc of titanium mesh is an effective treatment for this situation.

Purpose: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study. Experimental Design: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non–small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms. Results: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed. Conclusions: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network.

Purpose: Immune checkpoint blockade has shown remarkable efficacy, but in only a minority of patients with cancer, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in antitumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes. Experimental Design: We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We further validated our findings using in vitro coculture and in vivo syngeneic tumor growth assays. Results: We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti–PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T-cell–mediated killing. Man2a1 knockout enhanced response to anti–PD-L1 treatment and facilitated higher cytotoxic T-cell infiltration in tumors under anti–PD-L1 treatment. Furthermore, a pharmacologic inhibitor of MAN2A1, swainsonine, synergized with anti–PD-L1 in syngeneic melanoma and lung cancer models, whereas each treatment alone had little effect. Conclusions: Man2a1 loss renders cancer cells more susceptible to T-cell–mediated killing. Swainsonine synergizes with anti–PD-L1 in suppressing tumor growth. In light of the limited efficacy of anti–PD-L1 and failed phase II clinical trial on swainsonine, our study reveals a potential therapy combining the two to overcome tumor immune evasion. See related commentary by Bhat and Kabelitz, p. 5778

Due to their various biological activities that are beneficial to human health and antitumor effect, flavonoid compounds have attracted much attention in recent years. Hydrous icaritin (HICT) was such a flavonoid that can inhibit the growth of breast cancer and cancer stem cells. In order to overcome the insolubility problem, HICT was fabricated into nanorods (NRs) through anti-solvent precipitation in this paper using D-α tocopherol acid polyethylene glycol succinate and sodium oleate as a co-stabilizer meanwhile using the mixture of ethanol and acetone (1:2, v/v) as the organic solvent. The obtained HICT NRs showed an average particle size 222.0 nm with a small polydispersity index value of 0.124 and a high zeta potential of – 49.5 mV. HICT NRs could maintain similar particle size in various physiological medium and could be directly lyophilized without the addition of any cytoprotectants and then reconstituted into a colloidal system of similar size. The resultant HICT NRs had a high drug loading content of 55.6% and released HICT in a steady and constant pattern. MTT assay indicated NRs enhanced HICT’s antitumor activity to ninefold against MCF-7 breast carcinoma cells. In vivo studies demonstrated oral administration free HICT had almost no tumor inhibitory effect while HICT NRs showed a tumor inhibition rate of 47.8%. When intravenously injected, HICT NRs displayed similar therapeutic efficacy to paclitaxel injections (70.4% vs. 74.5%, TIR). This may be partly due to the high accumulation of the injected HICT NRs in tumor ranking only second to that in the liver but much higher than in other organs. These results demonstrated that HICT NRs could be a promising antitumor agent for the treatment of breast cancer in clinic.

BackgroundImmune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%–20% of patients and underpinnings of resistance remain undefined.MethodsStarting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models.ResultsAnti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1− (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions.ConclusionsThese data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.