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The acquisition of DNA damage is an early driving event in tumorigenesis. Premalignant lesions show activated DNA damage responses and inactivation of DNA damage checkpoints promotes malignant transformation. However, DNA damage is also a targetable vulnerability in cancer cells. This requires a detailed understanding of the cellular and molecular mechanisms governing DNA integrity. Here, we review current work on DNA damage in tumorigenesis. We discuss DNA double strand break repair, how repair pathways contribute to tumorigenesis, and how double strand breaks are linked to the tumor microenvironment. Next, we discuss the role of oncogenes in promoting DNA damage through replication stress. Finally, we discuss our current understanding on DNA damage in micronuclei and discuss therapies targeting these DNA damage pathways.

Heme oxygenase-1 (HO-1) is an inducible heme degradation enzyme that plays a cytoprotective role against various oxidative and inflammatory stresses. However, it has also been shown to exert an important role in cancer progression through a variety of mechanisms. Although transcription factors such as Nrf2 are involved in HO-1 regulation, the posttranslational modifications of HO-1 after oxidative insults and the underlying mechanisms remain unexplored. Here, we screened and identified that the deubiquitinase USP7 plays a key role in the control of redox homeostasis through promoting HO-1 deubiquitination and stabilization in hepatocytes. We used low-dose arsenic as a stress model which does not affect the transcriptional level of HO-1, and found that the interaction between USP7 and HO-1 is increased after arsenic exposure, leading to enhanced HO-1 expression and attenuated oxidative damages. Furthermore, HO-1 protein is ubiquitinated at K243 and subjected to degradation under resting conditions; whereas when after arsenic exposure, USP7 itself can be ubiquitinated at K476, thereafter promoting the binding between USP7 and HO-1, finally leading to enhanced HO-1 deubiquitination and protein accumulation. Moreover, depletion of USP7 and HO-1 inhibit liver tumor growth in vivo, and USP7 positively correlates with HO-1 protein level in clinical human hepatocellular carcinoma (HCC) specimens. In summary, our findings reveal a critical role of USP7 as a HO-1 deubiquitinating enzyme in the regulation of oxidative stresses, and suggest that USP7 inhibitor might be a potential therapeutic agent for treating HO-1 overexpressed liver cancers.

Effects of MET and STING signals on MET OE tumor after the intervention of MET inhibitor in vivo

Supplementary Methods

Baseline characteristics of patients

Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3′-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET.Significance:We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR–tyrosine kinase inhibitor resistant-related MET amplification.This article is highlighted in the In This Issue feature, p. 2659

Antibodies used for Western Blot experiments

Copy number variations in dabrafenib-persistent U266 single-cell clones as compared to baseline

Purpose:Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment.Experimental Design:Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis.Results:Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells.Conclusions:This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.

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Purpose:Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment.Experimental Design:Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis.Results:Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells.Conclusions:This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.

BRCA1-BARD1 heterodimers act in multiple steps during homologous recombination (HR) to ensure the prompt repair of DNA double strand breaks. Dysfunction of the BRCA1 pathway enhances the therapeutic efficiency of poly-(ADP-ribose) polymerase inhibitors (PARPi) in cancers, but the molecular mechanisms underlying this sensitization to PARPi are not fully understood. Here, we show that cancer cell sensitivity to PARPi is promoted by the ring between ring fingers (RBR) protein RNF19A. We demonstrate that RNF19A suppresses HR by ubiquitinating BARD1, which leads to dissociation of BRCA1-BARD1 complex and exposure of a nuclear export sequence in BARD1 that is otherwise masked by BRCA1, resulting in the export of BARD1 to the cytoplasm. We provide evidence that high RNF19A expression in breast cancer compromises HR and increases sensitivity to PARPi. We propose that RNF19A modulates the cancer cell response to PARPi by negatively regulating the BRCA1-BARD1 complex and inhibiting HR-mediated DNA repair., BRCA1 dysfunction sensitizes cancer cells to PARP inhibitors (PARPi) but the underlying mechanism is unclear. Here, the authors identify RNF19A as a determinant of PARPi sensitivity, showing that RNF19A ubiquitinates BARD1, negatively regulates the BRCA1-BARD1 complex, and inhibits homologous recombination.

The shieldin complex functions as the downstream effector of 53BP1-RIF1 to promote DNA double-strand break end-joining by restricting end resection. The SHLD2 subunit binds to single-stranded DNA ends and blocks end resection through OB-fold domains. Besides blocking end resection, it is unclear how the shieldin complex processes SHLD2-bound single-stranded DNA and promotes non-homologous end-joining. Here, we identify a downstream effector of the shieldin complex, ASTE1, as a structure-specific DNA endonuclease that specifically cleaves single-stranded DNA and 3' overhang DNA. ASTE1 localizes to DNA damage sites in a shieldin-dependent manner. Loss of ASTE1 impairs non-homologous end-joining, leads to hyper-resection and causes defective immunoglobulin class switch recombination. ASTE1 deficiency also causes resistance to poly(ADP-ribose) polymerase inhibitors in BRCA1-deficient cells owing to restoration of homologous recombination. These findings suggest that ASTE1-mediated 3' single-stranded DNA end cleavage contributes to the control of DSB repair choice by 53BP1, RIF1 and shieldin.

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice., DNA repair: tying up loose ends Carefully regulated enzymatic processing of the ends of DNA strands is essential for efficient replication and damage repair while also minimizing the risk of genomic instability. Replication and repair depend on a mechanism known as DNA resection, in which enzymes trim back double-stranded DNA ends to leave single-stranded overhangs. Zhenkun Lou and colleagues at the Mayo Clinic in Rochester, USA, have reviewed the various steps involved in the initiation and control of DNA resection. There are multiple different DNA repair processes, and the manner in which resection occurs can determine which of these processes subsequently takes place. The authors note that cancer cells rely heavily on these repair pathways to survive radiotherapy and chemotherapy, and highlight research opportunities that might reveal therapeutically useful vulnerabilities in the resection mechanism.

Human C-terminal binding protein (CtBP)–interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair., C-terminal binding protein (CtBP) interacting protein (CtIP) is a fundamental factor for the initiation of DNA end resection to initiate DNA repair. Here the authors reveal mechanistic insights into the regulation of CtIP via the deubiquitinase USP52.

IntroductionChildhood cancer is a leading cause of death. It is unclear whether the COVID-19 pandemic has impacted childhood cancer mortality. In this study, we aimed to establish all-cause mortality rates for childhood cancers during the COVID-19 pandemic and determine the factors associated with mortality.MethodsProspective cohort study in 109 institutions in 41 countries. Inclusion criteria: children ResultsAll-cause mortality was 3.4% (n=71/2084) at 30-day follow-up, 5.7% (n=113/1969) at 90-day follow-up and 13.0% (n=206/1581) at 12-month follow-up. The median time from diagnosis to multidisciplinary team (MDT) plan was longest in low-income countries (7 days, IQR 3–11). Multivariable analysis revealed several factors associated with 12-month mortality, including low-income (OR 6.99 (95% CI 2.49 to 19.68); pConclusionsChildren with cancer are more likely to die within 30 days if infected with SARS-CoV-2. However, timely treatment reduced odds of death. This report provides crucial information to balance the benefits of providing anticancer therapy against the risks of SARS-CoV-2 infection in children with cancer.

FoxM1 activates genes that regulate S-G2-M cell-cycle progression and, when overexpressed, is associated with poor clinical outcome in multiple cancers. Here we identify FoxM1 as a tumor suppressor in mice that, through its N-terminal domain, binds to and inhibits Ect2 to limit the activity of RhoA GTPase and its effector mDia1, a catalyst of cortical actin nucleation. FoxM1 insufficiency impedes centrosome movement through excessive cortical actin polymerization, thereby causing the formation of non-perpendicular mitotic spindles that missegregate chromosomes and drive tumorigenesis in mice. Importantly, low FOXM1 expression correlates with RhoA GTPase hyperactivity in multiple human cancer types, indicating that suppression of the newly discovered Ect2-RhoAmDia1 oncogenic axis by FoxM1 is clinically relevant. Furthermore, by dissecting the domain requirements through which FoxM1 inhibits Ect2 GEF activity, we provide mechanistic insight for the development of pharmacological approaches that target protumorigenic RhoA activity.

The cytidine deaminase APOBEC3B (A3B) is an endogenous inducer of somatic mutations and causes chromosomal instability by converting cytosine to uracil in single-stranded DNA. Therefore, identification of factors and mechanisms that mediate A3B expression will be helpful for developing therapeutic approaches to decrease DNA mutagenesis. Arsenic (As) is one well-known mutagen and carcinogen, but the mechanisms by which it induces mutations have not been fully elucidated. Herein, we show that A3B is upregulated and required for As-induced DNA damage and mutagenesis. We found that As treatment causes a decrease of N6-methyladenosine (m6A) modification near the stop codon of A3B, consequently increasing the stability of A3B mRNA. We further reveal that the demethylase FTO is responsible for As-reduced m6A modification of A3B, leading to increased A3B expression and DNA mutation rates in a manner dependent on the m6A reader YTHDF2. Our in vivo data also confirm that A3B is a downstream target of FTO in As-exposed lung tissues. In addition, FTO protein is highly expressed and positively correlates with the protein levels of A3B in tumor samples from human non-small cell lung cancer patients. These findings indicate a previously unrecognized role of A3B in As-triggered somatic mutation and might open new avenues to reduce DNA mutagenesis by targeting the FTO/m6A axis.

Interrogation of cell-free DNA (cfDNA) represents an emerging approach to non-invasively estimate disease burden in multiple myeloma (MM). Here, we examined low-pass whole genome sequencing (LPWGS) of cfDNA for its predictive value in relapsed/ refractory MM (RRMM). We observed that cfDNA positivity, defined as ≥10% tumor fraction by LPWGS, was associated with significantly shorter progression-free survival (PFS) in an exploratory test cohort of 16 patients who were actively treated on diverse regimens. We prospectively determined the predictive value of cfDNA in 86 samples from 45 RRMM patients treated with elotuzumab, pomalidomide, bortezomib, and dexamethasone in a phase II clinical trial (NCT02718833). PFS in patients with tumor-positive and -negative cfDNA after two cycles of treatment was 1.6 and 17.6 months, respectively (HR 7.6, P 0.0001). Multivariate hazard modelling confirmed cfDNA as independent risk factor (HR 96.6, P = 6.92e-05). While correlating with serum-free light chains and bone marrow, cfDNA additionally discriminated patients with poor PFS among those with the same response by IMWG criteria. In summary, detectability of MM-derived cfDNA, as a measure of substantial tumor burden with therapy, independently predicts poor PFS and may provide refinement for standard-of-care response parameters to identify patients with poor response to treatment earlier than is currently feasible.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Characterization of the recurrent genetic alterations in PDAC will yield improved understanding and therapies for this disease. Here, we report that PDAC patients with elevated expression of METTL16, one of the “writers” of RNA N6-methyladenosine (m6A) modification, may benefits from poly (ADP-ribose) polymerase (PARP) inhibitor treatment. Mechanistically, METTL16 interacts with MRE11 in an RNA-dependent manner; and, this interaction inhibits MRE11’s exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 at Ser419 within its C terminus, resulting in METTL16 conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression levels show increased sensitivity to PARP inhibitors, especially when combined with gemcitabine. Thus, our findings have revealed a role for METTL16 in homologous recombination repair and suggest that combination of PARP inhibitors with gemcitabine could be an effective treatment strategy for PDAC patients with high METTL16 expression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the 'writers' of RNA N

PURPOSE: Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment. EXPERIMENTAL DESIGN: Full-length single cell (sc)RNA-seq was conducted on three patients with BRAF-mutated myeloma and one healthy donor. We sequenced 1495 cells before, after one week and at clinical relapse to BRAF/ MEK inhibitor treatment. We developed an in vitro model of dabrafenib-resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K23ac ChIP-seq. Mitochondrial metabolism was characterized by metabolic flux analysis. RESULTS: Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in myeloma patients and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells. CONCLUSIONS: This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.

PARP inhibitors induce DNA lesions, the repair of which are highly dependent on homologous recombination (HR), and preferentially kill HR‐ deficient cancers. However, cancer cells have developed several mechanisms to transform HR and confer drug resistance to PARP inhibition. Therefore, there is a great clinical interest in exploring new therapies that induce HR deficiency (HRD), thereby sensitizing cancer cells to PARP inhibitors. Here, we found that GSK2578215A, a high‐selective and effective leucine‐rich repeat kinase 2 (LRRK2) inhibitor, or LRRK2 depletion suppresses HR preventing the recruitment of RAD51 to DNA damage sites through disruption of the interaction of RAD51 and BRCA2. Moreover, LRRK2 inhibition or depletion increases the susceptibility of ovarian cancer cells to Olaparib in vitro and in vivo. In clinical specimens, LRRK2 high expression is high related with advanced clinical characteristics and poor survival of ovarian cancer patients. All these findings indicate ovarian cancers expressing high levels of LRRK2 are more resistant to treatment potentially through promoting HR. Furthermore, combination treatment with an LRRK2 and PARP inhibitor may be a novel strategy to improve the effectiveness of LRRK2 expression ovarian cancers., LRRK2 inhibitor GSK2578215A suppresses HR and sensitizes ovarian cancer cells to PARP inhibitor. LRRK2 inhibition impedes the recruitment of RAD51 by disrupting the interaction of RAD51 and BRCA2. High expression of LRRK2 is associated with poor survival of ovarian cancer patients.

While there is extensive evidence for genetic variation as a basis for treatment resistance, other sources of variation result from cellular plasticity. Using multiple myeloma as an example of an incurable lymphoid malignancy, we show how cancer cells modulate lineage restriction, adapt their enhancer usage and employ cell-intrinsic diversity for survival and treatment escape. By using single-cell transcriptome and chromatin accessibility profiling, we show that distinct transcriptional states co-exist in individual cancer cells and that differential transcriptional regulon usage and enhancer rewiring underlie these alternative transcriptional states. We demonstrate that exposure to standard treatment further promotes transcriptional reprogramming and differential enhancer recruitment while simultaneously reducing developmental potential. Importantly, treatment generates a distinct complement of actionable immunotherapy targets, such as CXCR4, which can be exploited to overcome treatment resistance. Our studies therefore delineate how to transform the cellular plasticity that underlies drug resistance into immuno-oncologic therapeutic opportunities.

PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3′-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. Significance: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR–tyrosine kinase inhibitor resistant-related MET amplification. This article is highlighted in the In This Issue feature, p. 2659

The shieldin complex functions as the downstream effector of 53BP1-RIF1 to promote DNA double-strand break end-joining by restricting end resection. The SHLD2 subunit binds to single-stranded DNA ends and blocks end resection through OB-fold domains. Besides blocking end resection, it is unclear how the shieldin complex processes SHLD2-bound single-stranded DNA and promotes non-homologous end-joining. Here, we identify a downstream effector of the shieldin complex, ASTE1, as a structure-specific DNA endonuclease that specifically cleaves single-stranded DNA and 3' overhang DNA. ASTE1 localizes to DNA damage sites in a shieldin-dependent manner. Loss of ASTE1 impairs non-homologous end-joining, leads to hyper-resection and causes defective immunoglobulin class switch recombination. ASTE1 deficiency also causes resistance to poly(ADP-ribose) polymerase inhibitors in BRCA1-deficient cells owing to restoration of homologous recombination. These findings suggest that ASTE1-mediated 3' single-stranded DNA end cleavage contributes to the control of DSB repair choice by 53BP1, RIF1 and shieldin.

Introduction: Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming. Methods: 5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined. Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, 'scVelo' and 'CytoTRACE' were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using 'SCENIC'. Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models. Results: Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1. Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape. Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis. We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models. Conclusion: Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

The inflammatory responses in chronic airway diseases leading to emphysema are not fully defined. We hypothesised that lung eosinophilia contributes to airspace enlargement in a mouse model and to emphysema in patients with chronic obstructive pulmonary disease (COPD).A transgenic mouse model of chronic type 2 pulmonary inflammation (I5/hE2) was used to examine eosinophil-dependent mechanisms leading to airspace enlargement. Human sputum samples were collected for translational studies examining eosinophilia and matrix metalloprotease (MMP)-12 levels in patients with chronic airways disease.Airspace enlargement was identified in I5/hE2 mice and was dependent on eosinophils. Examination of I5/hE2 bronchoalveolar lavage identified elevated MMP-12, a mediator of emphysema. We showed,in vitro, that eosinophil-derived interleukin (IL)-13 promoted alveolar macrophage MMP-12 production. Airspace enlargement in I5/hE2 mice was dependent on MMP-12 and eosinophil-derived IL-4/13. Consistent with this, MMP-12 was elevated in patients with sputum eosinophilia and computed tomography evidence of emphysema, and also negatively correlated with forced expiratory volume in 1 s.A mouse model of chronic type 2 pulmonary inflammation exhibited airspace enlargement dependent on MMP-12 and eosinophil-derived IL-4/13. In chronic airways disease patients, lung eosinophilia was associated with elevated MMP-12 levels, which was a predictor of emphysema. These findings suggest an underappreciated mechanism by which eosinophils contribute to the pathologies associated with asthma and COPD.

Introduction: Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. A multitude of strategies preventing NOTCH1 cleavage and activation, such as Gamma-secretase inhibitors (GSIs) have been developed. Despite promising pre-clinical data, the rapid development of Notch1 inhibitor resistance in early clinical trials, prevented the translation of these inhibitors into the clinical setting. In previous work, our group demonstrated that T-ALL resistant to NOTCH1 inhibition carry altered epigenetic states conferring unique dependency on epigenetic modifiers, such as BRD4. The goal of this study was to study enhancer rewiring in Notch1 inhibitor resistant T-ALL in vivo and its relationship to apoptotic priming. Methods: After reaching 5% of circulating leukemic blasts, five established T-ALL PDX models with aberrant NOTCH1 expression were divided into to two treatment groups each (8 mice per group). 1 group received the Notch inhibitor DBZ (Dibenzazepine; 10 μM/kg intraperitoneal every other day) and the other group was treated with vehicle. Short-term effect of DBZ in vivo was assessed after 1 week of treatment, when 3 mice per group were sacrificed and leukemic blasts were isolated from spleen and bone marrow. The remaining 5 mice were monitored for disease burden (by flow cytometry staining for human CD45+) and followed for survival. After reaching moribund state, animals were sacrificed, spleens and bone marrows were collected and prepared for further analyses. To assess DBZ efficacy in vivo, the presence of active NOTCH1 (ICN1) in spleen and bone marrow was analyzed by Immunohistochemistry analysis (IHC). Enhancer landscapes were identified by chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) for Histone 3 Lysine 27 acetylation (H3K27ac). A custom computational pipeline that incorporates algorithms for demultiplexing, alignment, normalization, peak calling, and computation of signal intensities within peaks was used to call differential peaks and intersect with RNA-sequencing results. BH3 profiling was performed on leukemic blasts isolated from spleen to measure overall mitochondrial priming and to identify anti-apoptotic dependencies. Results: In four out of five T-ALL PDX models, IHC analysis of spleen and bone marrow demonstrated a drastic downregulation of active NOTCH1 upon DBZ treatment, validating the efficacy of the used inhibitor. Weak ICN1 staining that remained unchanged upon DBZ treatment, was observed in 1 of the models, resulting in the exclusion of this strain from further functional analysis. Survival analysis of the four T-ALL PDX models expressing ICN1, revealed the presence of two Notch inhibitor sensitive and two refractory strains. The latter strains developed DBZ resistance rapidly after starting treatment (less than 10 days). One sensitive strain eventually developed resistance, while the second showed long-term disease control. Transcriptional profiling (bulk RNA-seq) of Notch inhibitor refractory strains versus sensitive identified the intrinsic apoptotic pathway as one of the most deferentially deregulated GSEA signatures. H3K27ac ChIPseq analysis at pretreatment (baseline), showed increased signal intensity of H3K27ac peaks at BCL2 and MCL1 enhancers in the refractory strains compared to sensitive. Upon DBZ treatment, while the enhancer state in refractory T-ALL remained unchanged, in the sensitive strains the signal intensity of H3K27ac peaks within the BCL2 and MCL1 loci decreased. Mitochondrial BH3 profiling at baseline demonstrated BCL-2 dependency (measured via BAD peptide) in sensitive strains and MCL-1 dependency (measured via MS1 peptide) in refractory strains. Upon DBZ treatment, sensitive strains showed a decrease in BCL-2 dependency and compensatory switch to MCL1-dependency, while dependency profile remained unchanged in refractory T-ALL. Conclusions: Our results suggest that enhancer rewiring near anti-apoptotic genes is critical for Notch inhibitor resistance. Combining BH3 profiling with enhancer profiling may allow to predict drug responses in vivo and may contribute to the identification of novel therapeutic targets for combination therapy in resistant disease. Disclosures Letai: Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Weinstock:Celgene: Research Funding. Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding.

Introduction: Recent advancements in the treatment of multiple myeloma (MM) have centered on engaging the immune system to target multiple myeloma cells. Although these therapies are being combined with immunomodulatory imide drugs (IMiDs) and corticosteroids, the individual contributions of these drugs on the immune system of MM patients has not been examined in the upfront setting. In this study, we examined the peripheral blood immunophenotypes of newly diagnosed multiple myeloma (NDMM) patients receiving the IMiD lenalidomide with or without the corticosteroid dexamethasone. Methods: To characterize immunophenotypes, we utilized flow cytometry to profile white blood cell populations from 35 patients enrolled in a clinical trial testing the efficacy of lenalidomide with and without dexamethasone in NDMM (NCT00772915). In this trial, all patients were initiated on single-agent lenalidomide. Dexamethasone was initiated in patients that did not meet desirable responses or for disease progression. At each cycle, peripheral blood was stained with a 17-marker antibody panel against several immune lineages and functional surface markers. We grouped patients into two groups: 1) lenalidomide alone or 2) lenalidomide with dexamethasone according to their treatment regimen at each cycle timepoint. Results: First we confirmed anti-myeloma cell activity for both groups by measuring a steady decline in circulating plasma cells in both groups. Examining peripheral blood immunophenotypes showed an expected decrease in T cells and a smaller decrease in B cells in both groups of patients. Closer inspection of B cell populations revealed a switch towards a more immature B cell phenotype in both treatment groups. This was measured as a switch from CD19-CD20+ cells to CD19+CD20- B cells. Inspection of T cell subsets revealed that patients receiving single-agent lenalidomide had a sustained decrease in the levels of CD4+ T cells and increase in the levels of CD8+ T cells. This was seen in both naïve and regulatory T cells evidenced by a decrease in the CD4/CD8 ratio among CD28+ T cells as well as CD25+ T cells. Importantly, this alteration did not lead to sustained alterations in the overall level of CD25+ or CD28+ T cells, and the addition of dexamethasone reverses this trend. In addition to the effects seen on T and B cell numbers, we detected expansions of NK cell populations in patients receiving lenalidomide alone. This expansion is detected as an overall increase in CD56+ mononuclear cells with the majority of cells being CD56+CD3- cells. Conclusions: Our data show that lenalidomide and dexamethasone therapy have shared but distinct effects on peripheral blood immunophenotypes in NDMM. Both drugs alter B cells numbers and populations leading to an expansion of CD19+CD20- B cells. However, lenalidomide alone decreases the CD4/CD8 T cell ratio; and, lenalidomide more strongly expands NK cell populations. The addition of dexamethasone reverses this trend and leads to a restoration of the CD4/CD8 ratio. This suggests that lenalidomide without dexamethasone might be counterproductive in immunotherapies intended to recruit CD4+ T cells. Conversely, lenalidomide alone could increase the efficacy of immunotherapies dependent on NK cell recruitment such as antibody-dependent cellular cytotoxicity (ADCC). This information may benefit future investigations of immune responses in MM patients and improve the adoption of immunotherapies to MM patients. Figure Disclosures Kumar: Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Introduction: Despite recent advances in the treatment of multiple myeloma, responses may be short-lived and therapeutic resistance develops almost invariably. Non-genetic cellular plasticity and dedifferentiation have recently emerged as a basis for therapeutic resistance in cancer as cells acquire transcriptional states which no longer depend on the drug target. Therefore, a better understanding of plasticity and adaptive state changes in myeloma cells is critical to develop effective therapeutic approaches that can overcome drug resistance. Here we show that cellular plasticity, though frequently invoked as a basis for therapeutic resistance in cancer, can also lead to new therapeutic opportunities. Methods: To define transcriptional states in myeloma at a single cell level, we performed fluorescence activated cell sorting and full-length single-cell RNA sequencing. We assayed a total 6000 CD38+CD138+ plasma cells and CD45+ immune cells from the bone marrow of 8 patients with relapsed and refractory multiple myeloma (RRMM) before and after immuno-modulatory treatment on a clinical trial with elotuzumab, pomalidomide, bortezomib and dexamethasone (Elo-PVD; NCT02718833) and 2 healthy donors. Surface expression of selected markers was validated by flow cytometry. Results: Assessing pre-treatment samples, we discovered that the transcriptional states of single myeloma cells are highly distinct between individual patients, despite the presence of the same established genomic classifiers, such as t(11;14). Furthermore, distinct transcriptional states co-exist within individual patients, indicating there is substantial inter- and intra-individual heterogeneity. Transcriptional states diverge from normal plasma cells towards more immature cells, of the B lymphoid lineage, suggesting a substantial cellular plasticity. Notably, we detected co-expression of myeloid and lymphoid developmental programs in the same single cells. Interestingly, these altered differentiation states were associated with up-regulation of potential immunotherapeutic targets, such as CD20, CD19, and CD33, indicating that this plasticity may result in novel therapeutic vulnerabilities. To define gene-regulatory relationships, we identified a shared core regulatory network present in malignant and normal plasma cells with the active transcription factors XBP1, ATF4, and CREB3, suggesting that myeloma cells retain lineage-specific regulons. However, we further identified patient-specific regulons not detected in any of the mature immune cell populations assayed, such as TEAD4, ELF3 and SNAI1, illustrating an aberrant and promiscuous activation of transcriptional regulators in myeloma cells. Consistent with this finding, we observed an increased number of expressed genes in myeloma cells compared to normal plasma cells as well as an increase in single cell transcriptional entropy, measures that have been linked to cell potency in normal development and cancer. Comparison of pre- and post-treatment samples interestingly revealed a further increase in transcriptional diversity and signatures associated with stemness and developmental potential following treatment. Conclusions: In conclusion, we find that higher transcriptional diversity and activation of alternate gene regulatory programs facilitate the emergence of altered transcriptional states. Interestingly, these altered states are associated with up-regulation of putative immune-therapeutic targets in myeloma cells, thus providing novel therapeutic vulnerabilities. Disclosures Lipe: amgen: Research Funding; Celgene: Consultancy; amgen: Consultancy. O'Donnell:Celgene: Consultancy; Takeda: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Amgen: Consultancy. Munshi:Celgene: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Takeda: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder . Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding. OffLabel Disclosure: Samples for ancillary research were obtained in the context of a phase II clinical trial evaluating Elotuzumab, pomalidomide, bortezomib, dexamethasone The combination of elo-PVD is off label.

Introduction: Treatment of multiple myeloma (MM) has improved over the last decade. Although long-term survival is noted in some patients, emergence of resistant disease still prevents cures. The objective of this study was to define "liquid biopsy" parameters that identify patients who do not benefit from a particular treatment before relapse becomes evident by serological markers. Having such parameters at hand will potentially inform changes in treatment. Methods: Here, we apply low-pass whole genome sequencing to profile a uniform cohort of 45 relapsed and refractory MM (RRMM) patients who have been treated in a multicenter phase II trial evaluating the combination of elotuzumab, pomalidomide, bortezomib and dexamethasone (elo-PVd; NCT02718833). Peripheral blood plasma samples were acquired for circulating tumor DNA (ctDNA) evaluation at four different timepoints (screening, cycle 3 day 1 (C3D1), cycle 5 day 1 and end of treatment). The concentration, relative fraction and copy number profile of myeloma-derived ctDNA were determined across all timepoints. Results: At the time of this preliminary analysis, 17 patients (35%) continue on treatment whereas 28 patients (58%) have developed progressive disease (PD). Our data suggest that ctDNA levels at screening and C3D1 strongly correlate with progression-free survival (PFS). Patients with available follow-up samples (n=40) were stratified according to ctDNA levels at C3D1 of treatment. Patients with a residual ctDNA level Conclusions: These data indicate that "liquid biopsy" evaluation of ctDNA may refine prognostication and provide added predictive value over serological markers alone. While in the large majority of cases ctDNA has excellent concordance with M protein and SFLC for monitoring of MM disease progression, ctDNA may identify patients where relapse is imminent before it can be detected by serological parameters. This approach may therefore complement our framework for treatment decisions. Notably, this approach is highly scalable, cost-efficient and provides information about the clonal evolution of MM without the need for a bone marrow biopsy. Disclosures Yee: Adaptive: Consultancy; Amgen: Consultancy, Honoraria; Takeda: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Lipe:amgen: Research Funding; Celgene: Consultancy; amgen: Consultancy. O'Donnell:Sanofi: Consultancy; Amgen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Takeda: Consultancy. Munshi:Abbvie: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Oncopep: Consultancy. Richardson:Bristol-Myers Squibb: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Anderson:OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; Janssen: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board. Raje:Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Merck: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy. Lohr:T2 Biosystems: Honoraria; Celgene: Research Funding. OffLabel Disclosure: This abstract reports on the quadruple regimen elotuzumab, pomalidomide, bortezomib and dexamethasone which is not yet approved for the treatment of multiple myeloma in the United States.

Introduction: Constant clonal evolution and outgrowth of clones that harbor resistance mutations are likely explanations for the emergence of drug-resistant disease in multiple myeloma (MM). Activating mutations in BRAF, KRAS and NRAS have been suggested as potential therapeutic targets. In this study, we investigate resistance to BRAF inhibition in the context of BRAF-mutated MM which accounts for about 5-12% of all patients with relapsed/ refractory MM. Methods: Resistance to dabrafenib was modeled in vitro in the BRAF-mutated MM cell lines (MMCL) U266 (K601Nmut) and DP6 (BRAFV600Emut). Low-pass whole genome sequencing (LPWGS), RNA sequencing, ChIP sequencing and immunoblotting were performed for genomic, transcriptomic, epigenomic and molecular characterization. Functional validation was performed by genome editing using CRISPR/Cas9 technology. Results: Modeling of dabrafenib resistance in vitro revealed an initial decline of cell numbers, followed by a plateau phase and a gradual outgrowth of resistant cells after ~80 days of treatment. As expected, exposure of BRAFmut MMCL to dabrafenib led to initial downregulation of pERK and pMEK. At later timepoints, upregulation of pERK and pMEK was observed, suggesting that re-activation of the ERK/MEK pathway ultimately overcomes BRAF inhibition. This outgrowth was associated with highly distinct copy number profiles in each resistant clone, implying clonal selection with outgrowth of genetically resistant clones as one mechanism of drug resistance in MM. Additionally, we found that BRAF inhibition of BRAFmut MMCL promotes changes of the transcriptional circuitry that appears independent of clonal outgrowth of genetically resistant clones. These transcriptional changes were highly homogenous, occurred as early as 7-14 days after starting treatment and were associated with de-differentiation of MMCL into a more immature B lymphocytic phenotype. This phenotype was associated with greater mRNA expression of CD19 and CD81, as well as upregulation of the B-lymphocyte activation antigen B7-2 (CD86) and PI3K pathway genes. We next investigated if targeting the PI3K pathway and B7.2 can be exploited for effective killing of dabrafenib-resistant BRAFmut MM cells. Studies for the PI3Kδ inhibitor idelalisib in dabrafenib-persistent MMCL revealed higher sensitivity as compared to dabrafenib-naïve controls. Genome editing suggests a survival advantage for CD86WT as compared to CD86KO MMCL. Conclusions: Our data suggest that resistance to BRAF inhibition in vitro is mediated by two distinct mechanisms: 1) clonal outgrowth of genetically distinct resistant clones, and 2) transcriptional rewiring that leads to activation of alternative signaling pathways. The latter is characterized by changes in cellular differentiation and upregulation of PI3K and CD28/CD86 signaling. These concepts may provide a framework for revealing therapeutic vulnerabilities and to overcome drug resistance mediated by genetic heterogeneity in MM. Disclosures Munshi: Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Yee:Karyopharm: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Adaptive: Consultancy. Raje:Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria. OffLabel Disclosure: Dabrafenib is a potent inhibitor of BRAF mutated at codon 600 (BRAFV600). Here we explored the efficacy of dabrafenib a preclinical model of multiple myeloma cell lines with BRAFV600E and BRAFK601N mutations.

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

Eosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

Background Contact toxicant reactions are accompanied by localized skin inflammation and concomitant increases in site-specific itch responses. The role(s) of eosinophils in these reactions is poorly understood. However, previous studies have suggested that localized eosinophil-nerve interactions at sites of inflammation significantly alter tissue innervation. Objective To define a potential mechanistic link between eosinophils and neurosensory responses in the skin leading to itching. Methods BALB/cJ mice were exposed to different contact toxicants, identifying trimellitic anhydride (TMA) for further study on the basis of inducing a robust eosinophilia accompanied by degranulation. Subsequent studies using TMA were performed with wild type versus eosinophil-deficient PHIL mice, assessing edematous responses and remodeling events such as sensory nerve innervation of the skin and induced pathophysiological responses (ie, itching). Results Exposure to TMA, but not dinitrofluorobenzene, resulted in a robust eosinophil skin infiltrate accompanied by significant levels of degranulation. Follow-up studies using TMA with wild type versus eosinophil-deficient PHIL mice showed that the induced edematous responses and histopathology were, in part, causatively linked with the presence of eosinophils. Significantly, these data also demonstrated that eosinophil-mediated events correlated with a significant increase in substance P content of the cutaneous nerves and an accompanying increase in itching, both of which were abolished in the absence of eosinophils. Conclusions Eosinophil-mediated events following TMA contact toxicant reactions increase skin sensory nerve substance P and, in turn, increase itching responses. Thus, eosinophil-nerve interactions provide a potential mechanistic link between eosinophil-mediated events and neurosensory responses following exposure to some contact toxicants.