DNA Repair Signature Research Articles

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Abstract B032: Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers

Abstract We investigated the interplay between immunosuppressive TGFβ signaling and immunogenic DNA repair in response to immune checkpoint inhibition (ICI) in a new model of breast cancer. Transforming growth factor beta (TGFβ) is a canonical suppressor. Cancer cells evade TGFβ either by selectively overcoming TGFβ proliferative barrier or completely shutting down signaling. Loss of TGFβ signaling is accompanied by high immunosuppressive TGFβ activity. We have shown that loss of TGFβ is critical to genomic integrity. It endorses classical DNA repair and inhibits error-prone repair. We described a score, βAlt, that reports the TGFβ/DNA repair functional relationship. Pan-cancer analysis showed that high βAlt cancers, in which the TGFβ target signature is low and DNA repair signature is high, use error-prone DNA repair, are sensitive to chemoradiation and have a greater fraction of the genome altered compared to low βAlt cancers, which maintain TGFβ signaling. We established a novel murine tumor derived transplant model (mTDT) of triple-negative breast cancer, similar to a human PDX, in immunocompetent BALB/cJ mice. Transcriptomics analysis showed that mTDT range from high to low βAlt and from immune poor to immune rich, which was confirmed by multiplex immunstaining. Unsupervised clustering of mTDT based on βAlt score demonstrated that low βAlt was associated with immune rich tumors while high βAlt was associated with immune poor tumors. A similar relationship is evident in the TCGA breast cancers. We then asked how mTDT respond to ICI. Mice bearing mTDT were randomized to monotherapy consisting of single fraction 10 Gy radiation, anti-PD-L1 or TGFβ inhibition, dual or triple treatment. Neither anti-PD-L1 or TGFβ inhibition affected survival, alone or in combination, whereas radiation significantly increased survival. Unexpectedly immune poor, high βAlt mTDT were the most responsive. To evaluate biological correlates, mice were stratified as a function of treatment as responders, in which tumor growth was controlled for 7 days post-treatment, or non-responders, in which tumor growth was uncontrolled. Tumor infiltrating CD45+ cells increased (P=0.02) as did circulating CD8 T cells (P<0.001) in responders. Natural killer (NK) cells were also significantly expanded in responders (P<0.001). TGFβ broadly downregulates chemokine receptor expression necessary to recruit NK cells and impairs NK cell cytotoxic effector function. We depleted NK cells to test whether they were necessary for the pronounced response to triple treatment. NK depletion was completely abrogated response (P<0.00001) indicating NK cells can provoke an ICI response in high βAlt mammary tumors devoid of lymphocytes. This preclinical model and human breast cancer shows that loss of TGFβ signaling resulting in error-prone DNA damage response is strongly associated with immune poor tumors, and suggests that TGFβ inhibition can override NK cell dysfunction to create vulnerability to ICI and anti-tumor immunity. Citation Format: Jade Moore, Jim Gkantalis, Ines Guix, Colin Foster, Mary Helen Barcellos-Hoff. Compromised TGFβ signaling and DNA repair primes response to immune checkpoint inhibition by activating NK cells in immunologically cold breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B032.

Chromothripsis Orchestrates Leukemic Transformation in Blast Phase MPN through Targetable Amplification of DYRK1A

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question. To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp‘) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts ( p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status. Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3) Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mut TP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block. Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive toDYRK1A inhibitors, while healthy control CD34+ cells were unaffected Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in γ-H2AX foci after etoposide treatment or irradiation ( p<0.01 for both). A second mechanism of leukemogenesis emerged from geneset enrichment analyses, which suggested enhanced JAK-STAT signaling in chr21amp BPMPN cells and downregulation in the CRISPR DYRK1A KO context. We validated this by showing that DYRK1A overexpression activates and potentiates STAT5B transcriptional activity in a luciferase reporter assay. Finally, we noted that the STAT target BCL2 was selectively upregulated in chr21amp cells. BCL2 inhibition showed strong synergy with DYRK1A inhibitors for induction of BPMPN cell apoptosis (Bliss synergy score 15). Collectively, these findings define the chr21amp event as a novel prognostic biomarker in BPMPN. We pinpoint DYRK1A amplification as a central driver of genomic instability and exacerbated JAK-STAT signalling, for the first time linking chromothripsis to a specific druggable target ( Fig 1B).

RUNX1::RUNX1T1 Impairs the Differentiation Potential of Primary AML Cells

Acute myeloid leukemias (AMLs) are often driven by genomic translocations, of which t(8;21) is the most frequent. The resulting gene fusion RUNX1::RUNX1T has been shown in cell line and transgenic expression models to impede cell differentiation and promote leukemic self-renewal. However, it has remained unclear if and how RUNX1::RUNX1T1 influences leukemic propagation in patient-derived AML cells. To address the role of RUNX1::RUNX1T1 in the differentiation status and heterogeneity of patient-derived leukemic blasts, we developed an siRNA lipid nanoparticle (LNP) delivery system for the fusion transcript knockdown (Issa et al, Leukemia 2023). For efficient delivery to primary cells, the LNPs were functionalized with a Leu-Asp-Val (LDV) tripeptide binding the Very Late antigen-4 receptor on leukocytes. Primary AML cells were cultured and expanded for several weeks using a serum-free co-culture system with human bone marrow-derived mesenchymal stromal cells (MSCs) (Swart et al, Pharmaceutics 2023). Treatment of t(8;21) patient-derived xenograft (PDX) and primary bone marrow aspirates depleted RUNX1::RUNX1T1 transcript and protein, as determined by RT-qPCR and western blotting. We unraveled the transcriptional repercussions of RUNX1::RUNX1T1 knockdown using bulk RNAseq and ATACseq in a PDX and identified 1068 up- and 1521 downregulated genes upon RUNX1::RUNX1T1 knockdown, as compared to mismatched siRNA-treated cells (padj < 0.001, log2FC > 2). Gene set enrichment analysis revealed loss of self-renewal potential and enhanced myeloid differentiation. Similarly, ATACseq revealed an increased GMP/monocyte-like chromatin accessibility and a decreased HCS/MPP-like one upon RUNX1::RUNX1T1 knockdown (Figure 1). Surprisingly and in contrast to previous cell line results, RUNX1::RUNX1T1 depletion correlated with increased cell cycle and DNA repair signatures. These combined data demonstrate that RUNX1::RUNX1T1 perturbance promotes differentiation of leukemic stem cells towards granulocytic and monocytic lineages. Furthermore, they suggest that the initial stages of differentiation are linked to increased proliferation in line with the increased proliferation capacity of GMPs compared to more immature progenitor states. To investigate the impact of RUNX1::RUNX1T1 on leukemic blast populations at greater granularity, we knocked RUNX1::RUNX1T1 down in 2 primary bone marrow aspirates and 1 PDX and performed single-cell (sc) RNAseq. Notably, principal component analysis revealed that loss of RUNX1::RUNX1T1 shifted all leukemic populations, while hardly affecting normal hematopoietic cells or MSCs (Figure 2). In all samples, pseudotime analysis showed a more differentiated state of cells upon RUNX1::RUNX1T1 depletion. For cell type annotation, we projected our datasets onto a reference normal bone marrow dataset (van Galen et al, Cell 2019). This projection confirmed differentiation toward granulocyte-monocyte progenitors (GMPs), granulocytes and monocytes. Strikingly, the confidence of cell type prediction according to the reference normal bone marrow was significantly and substantially higher upon RUNX1::RUNX1T1 depletion than in cells with unimpaired fusion transcript level, suggesting restoration of normal myeloid differentiation. In addition to that, primary RUNX1::RUNX1T1 AML samples also contained a new, distinct cell subpopulation that emerged upon RUNX1::RUNX1T1 depletion. That subpopulation could not be annotated as resembling any normal bone marrow cell type. However, it expressed eosinophil marker genes such as RNASE3 and EPX. We, therefore, labeled this cell subpopulation as aberrant eosinophilic. This reveals RUNX1::RUNX1T1's role in disrupting differentiation towards multiple lineages. Our work illustrates a strong dependency of the differentiation potential of t(8;21) AML cells on their driver gene RUNX1::RUNX1T1 in an ex vivo setting with PDX or primary cells. We demonstrate that RUNX1::RUNX1T1 hijacks more than one differentiation trajectory of the AML blasts. Our findings also highlight siRNA-loaded LNPs as an innovative therapeutic approach for fusion gene-driven AML by directly targeting the fusion gene as the leukemic driver.

Multi-Dimensional Molecular and Tumor-Microenvironment Analysis of Classic Hodgkin Lymphoma

Introduction Classic Hodgkin Lymphoma (CHL) is currently classified into histological subtypes based on morphology and antigen marker expression. However, additional biological features might help guide treatment strategies and inform prognosis. Here, we aimed to uncover disease heterogeneity and biologic subtypes of CHL based on multi-dimensional molecular profiling ( Fig A) capturing somatic gene mutations, malignant cell expression phenotypes and altered tumor microenvironment (TME) architecture. METHODS We performed exome/targeted sequencing on flow-sorted or micro-dissected HRS cells from fresh-frozen CHL biopsies (n=116) of patients treated at BC Cancer. In addition, we constructed tissue microarrays (TMA) from this cohort on which we performed GeoMx® Whole Transcriptome Assay (WTA) to obtain gene expression profiles from CD30+ HRS cells and multiplexed imaging analyses using multi-colour immunofluorescence (mIF) to delineate the spatial TME ecosystem. Cox regression analysis was performed to identify outcome differences according to molecular features in CHL patients treated with ABVD-like chemotherapy (n=104). Results Mutational and copy number analyses identified known recurrent driver events including mutations and copy number changes in SOCS1, STAT6, TNFAIP3, B2M, REL and the PDL1/PDL2 locus. These mutational findings provided a statistically powered framework to investigate the association between genomic alterations with pathological and clinical disease parameters including outcomes. Mutations affecting BCL7A, TNFAIP3, and 2p15 (REL) amplifications were strongly enriched in EBV- CHL compared to EBV+ CHL. In line with “thymic mutations” reported in grey zone lymphoma, STAT6, GNA13, ITPKB, and TBL1XR1 mutations were significantly enriched in CHL with involvement of the anterior mediastinum. ZNF217, SPEN and CD58 mutations were significantly associated with progression-free survival (PFS) (all P < .01). When investigating outcome correlation according to age group, genomic alteration in STAT6 including both single nucleotide variants and STAT6 amplification, was the most significant feature associated with unfavorable PFS in patients <45 years of age ( P = .013). Alterations in the antigen recognition system, including loss of major histocompatibility complex (MHC) is one of the most studied mechanisms of immune escape in CHL. Mutations in CSF2RB, GNA13, TNFAIP3, and STAT6 were significantly associated with MHC-I loss. Strikingly, all cases harboring CSF2RB mutations (n=23) also showed loss of MHC-I. GeoMx analysis revealed that IFN-a and IFN-g pathways were significantly upregulated in MHC-I+ HRS cells. Mutations in HIST1H1E and BCL7A were significantly enriched in cases with loss of both MHC-I and MHC-II expression. To define mutational subtypes of CHL, we next applied non-negative matrix factorization (NMF) consensus clustering and discovered four robust subsets of tumors (clusters) using recurrent genomic events. Cluster1 (C1) is characterized by mutations in TNFAIP3, CSF2RB and GNA13, younger age, loss of MHC-I, and up-regulation of GCB and fatty acid metabolism signatures. C2 was associated with old age at initial diagnosis, EBV positivity and significant upregulation of the IFN-g pathway. C3 is characterized by REL and STAT6 amplification andexpression of a DNA repair signature. C4 includes samples from mostly younger patients with STAT6 and B2M mutations and high expression of CCL17 by HRS cells. MIF analyses further identified correlations between each mutational NMF cluster and TME composition ( Fig B), and in particular specific CD4+ T cell subsets. The TME of tumors was significantly enriched by C1: FOXP3+Tregs, C2: LAG3+CD4+ cells, and C3: PD1+CD4+ cells. C4 tumors showed no positive correlation with any of these immune cell compartments. C2 tumors were also significantly associated with the abundance of CD68+ macrophages. Conclusion Our multi-dimensional profiling approach enabled us to delineate molecular profiles of HRS cells that are linked to distinct TME patterns. These linkages have implications for current pathogenesis models, molecular subtyping of CHL, and identification of cellular vulnerabilities that might be therapeutically exploitable via targeting of HRS cell phenotypes and/or immune escape mechanisms.

Epithelioid Mesothelioma Patients with Very Long Survival Display Defects in DNA Repair.

DNA repair has an important role in malignant pleural mesothelioma (MPM) tumorigenesis and progression. Prognostic/predictive biomarkers for better management of MPM patients are needed. In the present manuscript, we analyzed the expression of more than 700 genes in a cohort of MPM patients to possibly find biomarkers correlated with survival. A total of 54 MPM patients, all with epithelioid histology, whose survival follow-up and formalin-fixed paraffin-embedded tumors were available, were included in the study. Gene expression profiles were evaluated using a Nanostring platform analyzing 760 genes involved in different cellular pathways. The percentages of proliferating tumor cells positive for RAD51 and BRCA1 foci were evaluated using an immunofluorescence assay, as a readout of homologous recombination repair status. Patient median survival time was 16.9 months, and based on this value, they were classified as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation in the DNA damage/repair expression score was observed in LS and VLS as compared to SS and VSS. These findings were validated by the lower number of both RAD51 and BRCA1-positive tumor cells in VLS as compared to VSS. The down-regulation of DNA repair signature in VLS was functionally validated by a lower % of RAD51 and BRCA1-positive tumor cells. If these data can be corroborated in a prospective trial, an easy, cost-effective test could be routinely used to better manage treatment in MPM patients.

Short-term molecular consequences of chromosome mis-segregation for genome stability

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

Abstract 839: YES1 is a novel druggable oncogene in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine subtype of lung cancer. This tumor is characterized by its aggressiveness, refractoriness to therapies, poor prognosis, and early dissemination. The first line of treatment consists of platinum-based chemotherapy, and although patients initially respond very well to this therapy, they promptly develop resistance. Currently, there are no targeted therapies approved for the treatment of SCLC patients. Here, we identified YES1, a non-receptor tyrosine kinase, as a novel targetable candidate with a relevant role in SCLC. In silico analysis revealed that YES1 is overexpressed in SCLC patients compared to non-malignant lung tissues. In a multi-institutional cohort of 80 SCLC patients, 31% of cases showed high YES1 protein expression and 26% of all specimens had YES1 gain/amplification (CNV≥2.5), analyzed by immunohistochemistry and FISH, respectively. In addition, high expression of YES1 predicted both lower overall survival (OS) and progression free survival (PFS) (n=80). In accordance, YES1 mRNA expression significantly correlated with YES1 CNV in SCLC cell lines. We knocked-down YES1 in two human SCLC cell lines (DMS53 and H209) with high expression and amplification of YES1. Depletion of YES1 almost totally abrogated cell proliferation and the formation of cell line-derived organoids in 3D. Accordingly, inhibition of YES1 arrested the cell cycle and promoted cell apoptosis in vitro. Consistent with these results, knockdown of YES1 promoted a dramatic reduction of subcutaneous tumor growth in both models with high levels of YES1, leading to a rate of partial regression of 54% and 23% complete regression. A metastasis assay, performed by intracardiac injection of H209 cells, showed that inhibition of YES1 led to complete disappearance of metastatic lung, leg and liver tumors in 4 out of 5 mice. Gene set enrichment analysis (GSEA) of RNA sequencing data performed with YES1-depleted DMS53 cells revealed that cells lacking YES1 had a downregulation in DNA replication and DNA repair signatures. We next explored the effect of pharmacological inhibition of YES1 using the multityrosine kinase inhibitor dasatinib and the selective YES1 inhibitor CH6953755. Dasatinib and CH6953755 effectively reduced cell proliferation in vitro at doses &amp;lt;1000 nM in the YES-high lines in contrast to the H69 cell line (low expression of YES1, &amp;gt;10µM). More interestingly, dasatinib and CH6953755 decreased DMS53, H209 and a high-YES1 PDX (YU-16)-derived organoid growth. Pharmacological inhibition of YES1 significantly reduced the tumor growth of high-YES1 models: DMS53 and H209 xenografts, and TP135 and YU-16 PDXs, in contrast to low-YES1 models (H69 xenograft and YU-34 PDX). Our results provide evidence that YES1 is a new druggable oncogenic target in SCLC and that its inhibition could be a promising therapeutic strategy for a subset of patients with high YES1 expression or copy number gain/amplification. Citation Format: Esther Redin, Eva M. Garrido-Martin, Karmele Valencia, Miriam Redrado, Jose Luis Solorzano, Rafael Carias, Franscisco Exposito, Diego Serrano, Nerea Otegui, Luis Paz Ares, Katerina Politi, Luis M. Montuenga, Alfonso Calvo. YES1 is a novel druggable oncogene in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 839.

HGG-50. Specific sensitivity of pediatric high-grade glioma with ATRX inactivation to PARP inhibitor combinations

Pediatric high-grade gliomas (pHGG) account for approximately 12% of pediatric brain tumors. Despite advances in molecular diagnosis and identification of discrete molecular subtypes, pHGG are the leading cause of cancer-related death in children. Thus, current research focuses on identifying novel therapeutic targets. Sequencing analyses across pediatric cancer types identified DNA repair perturbations as potentially targetable events in certain types of pediatric brain tumors. Herein, we investigated the potential of PARP inhibitors (PARPi), impeding the central role of PARP in DNA damage repair, in pHGG. We screened a patient-derived primary pHGG cell line panel (n=7) for their sensitivity towards 6 different PARPi (niraparib, olaparib, pamiparib, rucaparib, talazoparib, veliparib) using cell viability assays. Basal expression of DNA repair related proteins was assessed by immunoblot, and propidium iodide-based flow cytometry was used for cell cycle analysis. All pHGG were resistant towards single compound PARP inhibition. Interestingly, two H3F3A-G34R mutant pHGG models harboring inactivating ATRX mutations were characterized by elevated basal levels of pH2AX, suggesting increased stress resulting from DNA damage. Consequently, simultaneous targeting of PARP and other components of DNA repair in the respective models showed strong synergistic effects on cell viability, which was not observed to a comparable extent in other models such as BRAFV600E/TERT promotor mutant pHGG. Combination of talazoparib and irinotecan resulted in S-phase arrest. Within a precision oncology approach, we treated a 11-year-old child suffering from H3F3A-G34R mutant pHGG with ATRX mutation, that progressed during radiation, with niraparib and topotecan. The patient achieved partial remission and disease stabilization for 1 year. Taken together, PARPi combinations show potential for the treatment of pHGG with ATRX mutations. Currently, all cell models are characterized for DNA repair signatures by DNA sequencing. Further, in depth characterization of DNA damage responses upon concomitant PARP and topoisomerase inhibition in ATRX-mutated pHGG are ongoing.

Mechanisms of action and acquired resistance to atezolizumab plus nab-paclitaxel in metastatic triple-negative breast cancer (mTNBC).

1078 Background: In the IMpassion130 study (NCT02425891) first-line atezolizumab plus nab-paclitaxel (A+nP) provided clinical benefit compared with placebo plus nP (P+nP) in patients with mTNBC whose tumors were PD-L1+ (Schmid NEJM 2018). However, in many patients, disease that was initially controlled eventually progressed. The mechanism of action of A+nP and nP in the mTNBC tumor microenvironment (TME) and the biological changes associated with tumor progression with these therapies remain largely unknown. The goal of the current study was to evaluate biological changes in the TME induced by atezolizumab and nP early on treatment (OT) and at the time of progressive disease (PD) in IMpassion130. Methods: Paired tumor biopsies from IMpassion130 collected pre-treatment at baseline (BL), after 4 weeks OT, and at clinical PD were evaluated histologically for PD-L1 expression, CD8 content, stromal tumor infiltrating lymphocytes and immune phenotypes. RNA sequencing was also used to evaluate TNBC molecular subtypes and gene expression (hallmark gene sets, and immune cell and stromal gene signatures). Matched tumor pair samples from BL and PD were further analyzed by next-generation sequencing for genomic changes using the FoundationOne gene panel. Wilcoxon, Fisher, and McNemar’s tests were used for statistical analysis. Results: OT A+nP (n = 24 pairs), but not P+nP (n = 18 pairs) increased PD-L1 in both tumor-infiltrating immune cells and tumor cells, and increased frequency of immune-inflamed tumors. RNA-based signatures for A+nP showed an increase in lymphocytes (T-, B-, and NK cell), as well as IFN-α and IFN-γ responses, driven mainly by responders. While P+nP increased RNA-based stromal signatures (cancer-associated fibroblasts, pericytes, and angiogenesis) and epithelial mesenchymal transition, these changes were not observed with A+nP. OT A+nP and P+nP both reduced cell proliferation but only A+nP reduced metabolic pathways. At PD there was a significant reduction of RNA-based immune and stromal signatures in both A+nP (n = 59) and P+nP (n = 55) arms. Cell proliferation and DNA repair signatures were increased with A+nP but not P+nP. Evaluation of genomic changes suggested that both A+nP and P+nP increased tumor mutational burden (TMB), but only A+nP increased genomic scarring. At PD, the tumor immune phenotypes changed at PD with no directionality, while TNBC subtypes remained stable. Conclusions: A+nP boosted tumor immune inflammation and decreased tumor cell proliferation and metabolism in mTNBC patients, particularly in responders. Addition of atezolizumab prevented early stromal recruitment induced by nP. While decreased immune and stromal components and increased TMB were observed with both nP and A+nP, A+nP tumor escape was characterized by increased cell proliferation and DNA scarring. Clinical trial information: NCT02425891.

TAL1 cooperates with PI3K/AKT pathway activation in T-cell acute lymphoblastic leukemia.

TAL1 is ectopically expressed in about 30% of T-cell acute lymphoblastic leukemia (T-ALL) due to chromosomal rearrangements leading to the STIL-TAL1 fusion genes or due to non-coding mutations leading to a de novo enhancer driving TAL1 expression. Analysis of sequence data from T-ALL cases demonstrates a significant association between TAL1 expression and activating mutations of the PI3K-AKT pathway. We investigated the oncogenic function of TAL1 and the possible cooperation with PI3K-AKT pathway activation using isogenic pro-T-cell cultures ex vivo and in vivo leukemia models. We found that TAL1 on its own suppressed T-cell growth, in part by affecting apoptosis genes, while the combination with AKT pathway activation reduced apoptosis and was strongly driving cell proliferation ex vivo and leukemia development in vivo. As a consequence, we found that TAL1+AKTE17K transformed cells are more sensitive to PI3K-AKT pathway inhibition compared to AKTE17K transformed cells, related to the negative effect of TAL1 in the absence of activated PI3K-AKT signaling. We also found that both TAL1 and PI3K-AKT signaling increased the DNA-repair signature in T cells resulting in synergy between PARP and PI3K-AKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and have identified a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.

Validation of Anticorrelated TGFβ Signaling and Alternative End-Joining DNA Repair Signatures that Predict Response to Genotoxic Cancer Therapy.

Loss of TGFβ signaling increases error-prone alternative end-joining (alt-EJ) DNA repair. We previously translated this mechanistic relationship as TGFβ and alt-EJ gene expression signatures, which we showed are anticorrelated across cancer types. A score representing anticorrelation, βAlt, predicts patient outcome in response to genotoxic therapy. Here we sought to verify this biology in live specimens and additional datasets. Human head and neck squamous carcinoma (HNSC) explants were treated in vitro to test whether the signatures report TGFβ signaling, indicated by SMAD2 phosphorylation, and unrepaired DNA damage, indicated by persistent 53BP1 foci after irradiation or olaparib. A custom NanoString assay was implemented to analyze the signatures' expression in explants. Each signature gene was then weighted by its association with functional responses to define a modified score, βAltw, that was retested for association with response to genotoxic therapies in independent datasets. Most genes in each signature were positively correlated with the expected biological response in tumor explants. Anticorrelation of TGFβ and alt-EJ signatures measured by NanoString was confirmed in explants. βAltw was significantly (P < 0.001) better than βAlt in predicting overall survival in response to genotoxic therapy in The Cancer Genome Atlas (TCGA) pancancer patients and in independent HNSC and ovarian cancer patient datasets. Association of the TGFβ and alt-EJ signatures with their biological response validates TGFβ competency as a key mediator of DNA repair that can be readily assayed by gene expression. The predictive value of βAltw supports its development to assist in clinical decision making.

Single Cell Analyses Reveal a Non-Canonical EZH2 Activity As a Main Driver of Retinoic Acid Resistance in PLZF/Rara Leukemia

Resistance to treatment is due to the heterogeneity of the tumor which contains a subset of cancer cells that escape treatment and are responsible for the relapse. Acute Promyelocytic Leukemia (APL), the M3 subtype of AML, is a good model to illustrate these problematics. Indeed, APL driven by oncogenic fusion proteins such PML/RARA t(15;17) or PLZF/RARA t(11;17) behave differently to differentiation therapeutics. Both APLs differentiate in vivo upon Retinoic Acid (RA) treatment; however, while PML/RARA APL patients exhibit partial or complete remission, PLZF/RARA APL patients remain clinically resistant. In the present study we aim to decipher the transcriptional and epigenetic networks that is linked to t(11;17) APL resistance towards RA.We took advantage of the PLZF/RARA RA resistant murine APL model to catch relapse-initiating cell features and their vulnerabilities. By developing an integrative single-cell multi-omics analysis (scRNA-seq and scATAC-seq), we uncovered transcriptional and chromatin heterogeneity of the PLZF/RARA APL blasts. We highlighted a subset of cells insensitive to RA-induced differentiation with a strong DNA repair signature (“Rep” cluster) and exhibiting a fine tuned transcriptional network targeting the histone methyltransferase Ezh2. To validate the function of Ezh2 in APL physiology, we combined epigenomic studies with RA-treated and non-RA-treated bone marrow transplantation experiments. We revealed high Ezh2 activity that marks the relapse of RA-treated APL. However, targeting Ezh2 methyltransferase activity was not sufficient to achieve disease cure and, suggests an independent methyltransferase Ezh2 activity linked to RA resistance.These findings demonstrate the power of single-cell multi-omics integration to highlight path to sensitize therapy-resistant leukemia cells. In addition, our study uncovers a dual role of Ezh2 in APL and suggests that targeting non-canonical Ezh2 activity could be a new promising therapeutic approach for RA resistant APL. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

High-Dose Melphalan Significantly Increases Mutational Burden in Multiple Myeloma Cells at Relapse: Results from a Randomized Study in Multiple Myeloma

High-Dose Melphalan Significantly Increases Mutational Burden in Multiple Myeloma Cells at Relapse: Results from a Randomized Study in Multiple Myeloma

Immune- and tumor-intrinsic gene expression profiles of response or resistance to tislelizumab as monotherapy or in combination with chemotherapy in non-small cell lung cancer (NSCLC).

e15258 Background: Tislelizumab, an anti-PD-1 monoclonal antibody, showed clinical benefit for patients (pts) with NSCLC alone (NCT02407990, CTR20160872) and in combination with chemotherapy (NCT03432598). Gene expression profiles (GEP) associated with response and resistance to tislelizumab in these studies were assessed. Methods: The GEP of baseline tumor samples from 59 nonsquamous (NSQ) and 42 squamous (SQ) NSCLC pts treated with tislelizumab monotherapy (mono) as ≥1L treatment, and 16 NSQ and 21 SQ pts treated with tislelizumab plus chemotherapy (combo) as 1L treatment were assessed using the 1392-gene HTG GEP EdgeSeq panel. NSQ and SQ cohorts were analyzed separately due to distinct GEP features shown by PCA and t-SNE clustering. Results: Tislelizumab mono and combo showed antitumor activity in NSCLC (Table). In 80 biomarker-evaluable samples, inflamed tumor signatures (inflammatory GEP; antigen presentation GEP) were associated with longer overall survival (log-rank test, NSQ mono: P=0.04, 0.003; NSQ combo: P=0.05, 0.02; SQ combo: P=0.06, 0.06). Monotherapy non-responders (NRs) were clustered into 2 subgroups (NR1, NR2) with distinct GEPs. Compared with responders, NR1 had proliferation signatures (elevated cell cycle [CC] and DNA repair) in both NSQ ( P=0.2, 0.02) and SQ ( P=0.03, 0.4) cohorts, trending toward low inflamed tumor signatures. In NR pts receiving combo, CC and DNA repair signatures were not enriched, and high CC and DNA repair scores were observed in some SQ combo responders versus NRs ( P=0.2, 0.02). NR2 had higher M2 macrophage and Treg cell signatures versus responders in both NSQ and SQ mono, despite high inflamed tumor and low proliferation signatures. NR2 also had increased expression of genes related to immune regulation and angiogenesis, including PIK3CD, CCR2, CD244, IRAK3, and MAP4K1 ( P&lt;0.05) in NSQ, and PIK3CD, CCR2, CD40, CD163, MMP12, VEGFC, and TEK ( P&lt;0.05) in SQ. Conclusions: Clinical benefit in pts with NSCLC receiving tislelizumab (mono or combo) was associated with high inflamed tumor signatures, while elevated immune suppressive cell signatures may indicate resistance. High proliferation signatures were associated with resistance to monotherapy, but not to combination therapy. Both immune- and tumor-intrinsic factors may be considered for validation in future clinical trials. [Table: see text]

Efficacy and pharmacodynamics of niraparib in BRCA-mutant and wild-type intracranial triple-negative breast cancer murine models.

BackgroundDespite the poor prognosis of triple-negative breast cancer (TNBC) brain metastases, there are no approved systemic therapies. We explored the DNA-damaging poly(ADP-ribose) polymerase inhibitor (PARPi) niraparib in intracranial mouse models of breast cancer susceptibility protein (BRCA)-mutant TNBC.MethodsMice bearing intracranial human-derived TNBC cell lines (SUM149, MDA-MB-231Br, or MDA-MB-436) were treated with niraparib and monitored for survival; intracranial tissues were analyzed for PAR levels and niraparib concentration by mass spectrometry. RNASeq data of primary breast cancers using The Cancer Genome Atlas were analyzed for DNA damage signatures. Combined RAD51 and PARP inhibition in TNBC cell lines was assessed in vitro by colony-forming assays.ResultsDaily niraparib increased median survival and decreased tumor burden in the BRCA-mutant MDA-MB-436 model, but not in the BRCA-mutant SUM149 or BRCA-wild-type MDA-MB-231Br models despite high concentrations in intracranial tumors. RAD51 inhibitor B02 was shown to sensitize all cell lines to PARP inhibition (PARPi). In the analysis of BRCA-mutant primary human TNBCs, gene expression predictors of PARPi sensitivity and DNA repair signatures demonstrate widespread heterogeneity, which may explain the differential response to PARPi. Interestingly, these signatures are significantly correlated to RAD51 expression including PARPi sensitivity (R2 = 0.602, R2= 0.758).ConclusionsNiraparib penetrates intracranial tumor tissues in mouse models of TNBC with impressive single-agent efficacy in BRCA-mutant MDA-MB-436. Clinical evaluation of niraparib to treat TNBC brain metastases, an unmet clinical need desperate for improved therapies, is warranted. Further compromising DNA repair through RAD51 inhibition may further augment TNBC’s response to PARPi.

Age-Dependent Protective Effect of Selenium against UVA Irradiation in Primary Human Keratinocytes and the Associated DNA Repair Signature.

Few studies have focused on the protective role of selenium (Se) against skin aging and photoaging even though selenoproteins are essential for keratinocyte function and skin development. To the best of our knowledge, the impact of Se supplementation on skin cells from elderly and young donors has not been reported. Therefore, the main objective of our study was to evaluate the effects of Se supplementation on skin keratinocytes at baseline and after exposure to ultraviolet A (UVA) irradiation. Low doses of Se (30 nM) were very potently protective against UVA-induced cytotoxicity in young keratinocytes, whereas the protection efficiency of Se in old keratinocytes required higher concentrations (240 nM). Additionally, the DNA repair ability of the old keratinocytes drastically decreased compared with that of the young keratinocytes at baseline and after the UVA exposure. The Se supplementation significantly enhanced the DNA repair of 8-oxoguanine (8oxoG) only in the keratinocytes isolated from young donors. Therefore, aged keratinocytes have an increased vulnerability to oxidative DNA damage, and the Se needs in the elderly should be considered. Strengthening DNA repair activities with Se supplementation may represent a new strategy to combat aging and skin photoaging.

Abstract 1428: Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer

Abstract Resistance to chemotherapy poses a major clinical problem in the treatment of ovarian cancer. The efficacy of platinum- based chemotherapy relies on defective DNA repair mechanisms, and increased expression of homologous recombination (HR)/DNA repair genes in primary tumor samples is associated with improved chemoresponse. However, the effects of neo-adjuvant chemotherapy (NACT) on the expression DNA repair genes in cancer cells are poorly understood. In addition, high-grade serous ovarian cancers (HGSOCs) display significant inter- and intratumoral heterogeneity, and the identification of mechanisms of drug resistance has been hampered by the lack of data at the single cell resolution. We collected 34 primary and 20 interval tumor samples from 15 patients treated for HGSOC at the Turku University Hospital, Finland. Tumor tissue total RNA was sequenced with Illumina HiSeq. For this study, we selected 363 genes selected due to their validated role in DNA repair pathways and compared their expression profiles between primary and interval samples. The treatment naïve primary tumor samples highly expressed genes related to HR, such as BRCA2, RAD54, PARPBP, FANCD2, and POLQ. Whereas after NACT the interval samples highly expressed genes regulating cell cycle, inflammatory response, and the drug efflux pump ABCB1. Moreover, higher expression of BRCA2 or POLQ in the interval samples correlated with poor primary therapy outcome (p&amp;lt;0.01). To reveal the transcriptomics landscapes of treatment naïve and NACT treated HGSOCs at the single cell level, we performed single cell mRNA sequencing of primary and interval samples using the Fluidigm C1 Platform. Unsupervised clustering revealed eight distinct cell subpopulations, of which two clusters were annotated HGSOC cells based on enriched expression of cancer specific genes. In global gene expression analysis of 98 HGSOC cells, the most differentially expressed genes in primary HGSOC cells were heat shock proteins reflecting active protein synthesis. By contrast, the cancer cells from interval samples highly expressed genes regulating the cell cycle and immune response. Of the DNA repair pathway genes, the interval samples expressed lower levels of HES1, a transcription factor that is known to promote cancer progression and stemness. The herein elucidated DNA repair pathway transcriptomics landscape at single-cell and whole tumor level provides novel targets for overcoming chemoresistance in HGSOC. Citation Format: Anniina Farkkila, Kaiyang Zhang, Katja Kaipio, Tarja Lamminen, Rainer Lehtonen, Johanna Hynninen, Seija Grénman, Olli Carpén, Sampsa Hautaniemi, Anna Vähärautio. Single-cell transcriptomics reveals differential DNA repair signatures after chemotherapy in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2017-1428

Dynamic process of circulating tumour DNA (ctDNA) profiling as a predictor of efficacy of pemetrexed combined platinum chemotherapy in advanced lung adenocarcinoma.

e23048 Background: Pemetrexed combined platinum is recommended as first line chemotherapy regimen for non-squamous non-small cell lung cancer. However, the efficacy is variable for individuals. Methods: We conducted a prospective study with 52 consecutive patients enrolled who were histologically confirmed advanced or metastatic lung adenocarcinoma, of whom, 32 had their plasma samples collected both baseline and follow-up when efficacy evaluation. We used a next-generation sequencing to assess the dynamic change of ctDNA across 87 plasma samples. Results: 34 blood samples from 61.5% of patients demonstrated smoking status is an infavorable factor to drug efficacy ( p= 0.02, fisher’s exact test, two-sided). Simultaneously, we found positive correlation between decreased mutation burden and chemotherapy sensitivity (the mean mutation rates of pre chemotherapy, PD, PR, SD and SDA were 4.5, 6.3, 2.1, 1.6 and 2.4, respectively, p= 0.004, one way ANOVA test, two-sided).The mean mutation rates with T &gt; G substitution mutation of pre and post chemotherapy were 1.5 and 0.6 ( p= 0.014, Mann-Whitney U test, two-sided). 34.4% (11/32) individuals harbor those mutation pre chemotherapy, 6 out of them were characterized by DNA repair signatures, of which 83.3% (5/6) cases were positively related to platinum sensitivity. In addition, T &gt; G substitution was still detected from 5 patients at cycle 2, 60% (3/5) individuals were characterized by DNA repair signatures and 2 out of them had better platinum-based chemotherapy response.Neoplastic cells carrying a specific set of somatic mutations were sensitive to platinum treatment, including USP9X (pre vs post chemotherapy: 10.3% vs 0%), ZRSR2 (pre vs post chemotherapy: 6.9% vs 0%) and PIK3C2G (pre vs post chemotherapy: 10.3% vs 0%). Nonetheless, persistence of gene ROBO2 (pre vs post chemotherapy: 10.3% vs 4.6%) may be correlated with tumorigenesis. Conclusions: Dynamic monitoring of ctDNA mutation status may be contributed to advanced or metastatic lung adenocarcinoma patients stratification and prediction for pemetrexed combined platinum as first line chemotherapy regimen.

Abstract 306: DNA Repair Enzyme Signature of a cohort of 100 healthy individuals using peripheral blood mononuclear cells: In the search of cancer susceptibility biomarkers

Abstract It is well established that mutations and combinations of polymorphisms in DNA Repair genes contribute to cancer occurrence. However the relationship between genotypes and cancer is not straightforward. As a matter of fact, individuals’ cancer susceptibility also depends on their genotoxic exposure history, age and lifestyle. In this context, functional DNA-repair tests better account for DNA Repair complexity and constitute powerful strategies to characterize effective individuals DNA Repair capacity. Currently available tools interrogate very limited aspects of DNA Repair mechanism and more informative assays are urgently needed. In humans Base Excision Repair (BER) involves several glycosylases that have the ability to recognize and cleave a set of small base lesions resulting from oxidation, alkylation, or deamination of normal bases, with the help of an AP endonuclease (APE1) that removes abasic sites. To precisely characterize the BER pathway and offer better chance to identify individuals with altered DNA repair capacities, we developed a comprehensive approach that quantifies simultaneously several BER repair enzymes in cell extracts. This multiplexed biochip version of the well-known oligonucleotide cleavage assay was implemented to characterize the DNA Repair Enzyme Signature of a cohort of 100 healthy individuals (50% males and 50% females, ranging in age from 18 to 70 years). All were non-smokers. Two 8 mL blood samples were taken the first day and another one one week apart. Whole cell extracts were prepared from the subjects Peripheral Blood Mononuclear Cells (PBMCs) and protein concentration was determined. Assay robustness was assessed and the inter- and intra-individual BER variability was also determined. We observed a large inter-individual variability in DNA repair capacities. We demonstrated that there was a gender-specific impact of aging on certain DNA Repair activities. Thanks to the multiplexed approach, using hierarchical clustering and principal component analysis, we gained new information on the different glycosylase activities co-regulations and assigned the samples to distinct subpopulation clusters characterized by their DNA Repair Signature. Here again, males and females exhibited different features. We demonstrated that our powerful assay is robust, can be implemented on a large scale and could be used for population studies to identify biomarkers of exposure and biomarkers of cancer susceptibility. We thank GRAVIT and CEA program Technologies for Health for financial support. Citation Format: Sylvie Sauvaigo, Lise Jacqueroux, Benoit Pitiot, Sylvain Caillat, Nicolas Gonnet, Jean-Luc Cracowski. DNA Repair Enzyme Signature of a cohort of 100 healthy individuals using peripheral blood mononuclear cells: In the search of cancer susceptibility biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2014-306