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Drug discovery currently focuses on identifying new druggable targets and drug repurposing. Here, we illustrate a third domain of drug discovery: the dimensionality of treatment regimens. We formulate a new schema called ‘Manifold Medicine’, in which disease states are described by vectorial positions on several body-wide axes. Thus, pathological states are represented by multidimensional ‘vectors’ that traverse the body-wide axes. We then delineate the manifold nature of drug action to provide a strategy for designing manifold drug cocktails by design using state-of-the-art biomedical and technological innovations. Manifold Medicine offers a roadmap for translating knowledge gained from next-generation technologies into individualized clinical practice.

Objective. To describe molecular and clinical characteristics of patients with high-grade recurrent ovarian carcinoma (HGOC) who had long-term responses to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib. Methods. This post hoc analysis pooled patients from Study 10 (NCT01482715; Parts 2A and 2B; n = 54) and ARIEL2 (NCT01891344; Parts 1 and 2; n = 491). Patients with investigator-assessed complete or partial response per RECIST were classified based on duration of response (DOR): long (≥1 year), intermediate (6 months to

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved. In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages. In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic “scarring,” indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy. As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy. Significance: Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.

Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

Qiuju Liu,1 Brittny Major,2 Jennifer Le-Rademacher,2 Aref A Al-Kali,3 Hassan Alkhateeb,3 Kebede Begna,3 Michelle A Elliott,3 Naseema Gangat,3 William J Hogan,3 C Christopher Hook,3 Scott H Kaufmann,3 Animesh Pardanani,3 Mrinal S Patnaik,3 Ayalew Tefferi,3 Alexandra P Wolanskyj-Spinner,3 Wei Wei,4 Mark R Litzow3 1Department of Cancer Center, Division of Hematology, Jilin University, Changchun, Jilin, People’s Republic of China; 2Department of Health Sciences Research, Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA; 3Department of Internal Medicine, Division of Hematology, Mayo Clinic, Rochester, MN, USA; 4Department of Urology, Second Division, Jilin University, Changchun, Jilin, People’s Republic of ChinaCorrespondence: Mark R LitzowDivision of Hematology, Mayo Clinic, 200 First St, Rochester, MN 55902, USATel +1 507-284-2511Email litzow.mark@mayo.eduIntroduction: Obesity is a worldwide problem that is related to cardiac disease, thrombosis and cancer. However, little is known about the impact of obesity on the outcomes of adult acute lymphoblastic leukemia (ALL) patients.Methods: We retrospectively evaluated a cohort of 154 newly diagnosed adult ALL patients between 1994 and 2011 at Mayo Clinic (Rochester). According to the World Health Organization (WHO) international BMI classification, patients were stratified as underweight, normal weight, overweight, and obese. For some analyses, patients were also stratified according to a two-sided non-obese or obese classification.Results: The median follow-up time was 8.37 years. Obese patients were more likely to be women (p=0.024) and ≥ 60 years old (p=0.003). Five-year mortality rates were higher in obese patients than non-obese [HR 95% CI: 1.60 (1.03– 2.50) p=0.035]. This was also the case in subgroup analysis among T-cell patients although the number of patients was small [HR 95% CI: 5.42 (1.84– 15.98) p< 0.001]. There was no difference in mortality among the B-cell patients. After adjusting for baseline variables, the difference in mortality remained in several models. There was no difference in EFS or cumulative incidence of relapse rates between obese and non-obese patients among the overall population.Discussion: In conclusion, our study suggests that adult ALL patients with obesity have lower survival rates, especially in T-cell ALL.Keywords: acute lymphoblastic leukemia, obesity, overweight, B cell, T cell

Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Purpose:To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs.Experimental Design:Matched case–control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays.Results:Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (P = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (P = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient CYP19A1−/− T47D breast cancer cell line.Conclusions:This matched case–control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

In this issue of Cell Chemical Biology, Harvey et al. (2020) identify 4E14, a sulfhydryl-containing N-acetyltryptophan analog that selectively disrupts binding to the previously undruggable anti-apoptotic BCL2 paralog BFL1, and elucidate a BFL1 conformational change that facilitates 4E14 interaction. These results provide insight that will accelerate development of BFL1 inhibitors.

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies., DNA-protein crosslinks represent obstacles on genomic DNA that can hamper progression of replication forks. Here, the authors reveal that FAM111A, a PCNA-interacting protein, plays part in mitigating the effect of protein obstacles on replication forks.

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60–3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. Significance: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Cancer stem cells (CSCs) represent a small fraction of the total cancer cell population, yet they are thought to drive disease propagation, therapy resistance and relapse. Like healthy stem cells, CSCs possess the ability to self-renew and differentiate. These stemness phenotypes of CSCs rely on multiple molecular cues, including signaling pathways (for example, WNT, Notch and Hedgehog), cell surface molecules that interact with cellular niche components, and microenvironmental interactions with immune cells. Despite the importance of understanding CSC biology, our knowledge of how neighboring immune and tumor cell populations collectively shape CSC stemness is incomplete. Here, we provide a systems biology perspective on the crucial roles of cellular population identification and dissection of cell regulatory states. By reviewing state-of-the-art single-cell technologies, we show how innovative systems-based analysis enables a deeper understanding of the stemness of the tumor niche and the influence of intratumoral cancer cell and immune cell compositions. We also summarize strategies for refining CSC systems biology, and the potential role of this approach in the development of improved anticancer treatments. Because CSCs are amenable to cellular transitions, we envision how systems pharmacology can become a major engine for discovery of novel targets and drug candidates that can modulate state transitions for tumor cell reprogramming. Our aim is to provide deeper insights into cancer stemness from a systems perspective. We believe this approach has great potential to guide the development of more effective personalized cancer therapies that can prevent CSC-mediated relapse.

Background: Ovarian cancer ranks fifth in cancer deaths among women. The gold standard of care for advanced disease includes cytoreductive surgery along with adjuvant or neoadjuvant platinum/taxane-based chemotherapy. Although response to this treatment is high, chemotherapy-induced nausea and vomiting due to platinum agents and allergic reactions due to both agents are common. For nearly half a century, the glucocorticoid (GC) dexamethasone (DEX) has been administered as part of supportive care. However, there have periodically been questions about the possible deleterious effects of DEX on chemotherapy response, especially in solid tumors. Methods: Platinum-sensitive, glucocorticoid receptor (GRα) expressing ovarian cancer cells were exposed to DEX prior to chemotherapy and clonogenic survival was assessed. The effects of DEX on cell cycle and apoptosis were assessed by flow cytometry. Changes in the tumor cell transcriptome after acute DEX treatment were determined by RNA sequencing. Mice bearing orthotopic ovarian cancer patient-derived xenografts (PDXs) were treated with chemotherapy ± DEX to assess changes in chemotherapy response in vivo. Findings: DEX pre-treatment decreased platinum cytotoxicity in ovarian cancer cell lines in vitro and diminished platinum response in some PDXs analyzed for chemotherapy response ex vivo. RNA-seq analysis revealed changes in transcripts encoding proteins involved in platinum detoxification and TNFα pro-survival signaling. In ovarian cancer PDXs, DEX decreased platinum response in one model and had limited impact on both platinum and platinum/taxane chemotherapy in three distinct studies. Interpretation: DEX appears to modulate chemotherapy response in some settings in vitro and ex-vivo. The impact of DEX on chemotherapy response in vivo appears to be limited. However, further investigation of this question in vivo in non-immunosuppressed murine models appears warranted. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Cordelia D. Mcgehee, Sarah Finstuen-Magro, Jamison VanBlaricom, Paula A. Schneider, Kevin L. Peterson, Ethan P. Heinzen, Melissa C. Larson, Xiaonon Hou, Ann L. Oberg, Saravut John Weroha, Scott H. Kaufmann. Impact of dexamethasone on chemotherapy response in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5058.

Ovarian cancer is one of the most common gynecologic malignancies, with high-grade serous carcinoma (HGSC) as the most frequent and deadliest type of ovarian cancer (OC). With the recent advances in cancer epigenomic profiling, it has become clear that intra-tumor heterogeneity driven by genetic and epigenetic factors may be the basis of resistance, where current treatments could efficiently target sub-populations of cells while a sub-group may be resistant, or prone to develop resistance. With epigenomic profiling and single cell sequencing technology, we aim to characterize cell signatures that translate as prognostic markers and novel drug targets. We have mapped the epigenomic landscape of HGSC in 12 patient-derived xenografts (PDX), and their metastatic counterparts (circulating tumor cells and ascites present in the PDX models) using an assay for transposase-accessible chromatin (or ATAC-seq) and compared it with normal fallopian tube organoids to identify tumor and metastatic-specific regulatory elements. We also performed RNA-seq to validate potential transcriptional dependencies associated with resistance to chemotherapy. In order to identify potentially chemotherapy-resistant subpopulations and further validate the activity of some of the transcription factors that can be driving resistance, we performed single cell multiomic profiling using the 10x Multiome sequencing platform (scRNA+scATAC from the same nuclei) in HGSC patient samples and PDX. Finally, we performed immunohistochemistry of patient tissue slides for target markers and correlated it with overall survival in OC patients. As previously suggested, a subset of cancer clonal cells can drive treatment resistance and metastasis and result in poor patient outcomes. Data derived from PDX ATAC-seq showed a cancer stem cell signature driven by SOX2 in metastatic samples compared to the paired PDX primary tumor, suggesting a role for SOX2 in disease progression. Interestingly, SOX2 expression in epithelial cells in the Multiome analysis is more patient-specific than other stem cell markers, like CD133. We also observed that SOX2 expression negatively correlated with prognosis. These data, taken together, point out the promising role of SOX2 as a prognostic and targetable marker for HGSC. Citation Format: Leticia Assad Maia Sandoval, Wazim Mohammed Ismail, Amelia Mazzone, Amik Munankarmy, Jagneet Kaur, Tsering Tashi, Xiaonan Hou, Nagarajan Kannan, Ann Oberg, Scott H. Kaufmann, Saravut John Weroha, Alexandre Gaspar-Maia. Single cell multiomic profiling of high-grade serous carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4724.

BCL2 family proteins play an essential role in controlling mitochondrial outer membrane permeabilization which is the fatal and usually irreversible step of the mitochondrial apoptotic pathway. Moreover, BCL2 family proteins are also involved in the regulations of other cellular processes, such as autophagy, mitochondrial fission and fusion, and endoplasmic reticulum stress. Three proteins, BAK, BAX, and BOK, are the effector BCL2 family proteins that directly permeabilize the mitochondrial outer membrane while the antiapoptotic BCL2 family members and the BH3-only subfamily proteins regulate this process through protein–protein interactions. BCL2 family proteins are known to be important in the development of several diseases, especially cancer. Thus, much effort has gone into the development of drugs targeting BCL2 family proteins to treat cancer; developed agents include antisense oligonucleotides, modified peptides, and small molecules. Some of these efforts have been quite successful, with several candidates tested in clinical trials. Moreover, ABT-199 (venetoclax) was approved by the United States Food and Drug Administration to treat relapsed, chromosome 17p-deleted chronic lymphocytic leukemia in 2016, providing a huge inspiration for this field. In this chapter, we summarize the recent advances in BCL2 family studies, review the latest developments and clinical studies of drug candidates to target BCL2 family proteins, and discuss future directions of this class of antineoplastic agents.

How BAK and BAX induce mitochondrial outer membrane (MOM) permeabilization (MOMP) during apoptosis is incompletely understood. Here we have used molecular dynamics simulations, surface plasmon resonance, and assays for membrane permeabilization in vitro and in vivo to assess the structure and function of selected BAK subdomains and their derivatives. Results of these studies demonstrate that BAK helical regions α5 and α6 bind the MOM lipid cardiolipin. While individual peptides corresponding to these helical regions lack the full biological activity of BAK, tandem peptides corresponding to α4-α5, α5-α6, or α6-α7/8 can localize exogenous proteins to mitochondria, permeabilize liposomes composed of MOM lipids, and cause MOMP in the absence of the remainder of the BAK protein. Importantly, the ability of these tandem helices to induce MOMP under cell-free conditions is diminished by mutations that disrupt the U-shaped helix-turn-helix structure of the tandem peptides or decrease their lipid binding. Likewise, BAK-induced apoptosis in intact cells is diminished by CLS1 gene interruption, which decreases mitochondrial cardiolipin content, or by BAK mutations that disrupt the U-shaped tandem peptide structure or diminish lipid binding. Collectively, these results suggest that BAK structural rearrangements during apoptosis might mobilize helices involved in specific protein-lipid interactions that are critical for MOMP.

Simple Summary Poly(ADP-ribose) polymerase (PARP) inhibitors, which are medications approved to treat various solid tumors, including breast, prostate, ovarian, and prostate cancers, are being examined in hematological malignancies. This review summarizes the potential role of PARP inhibitors in the treatment of myeloid diseases, particularly acute myeloid leukemia (AML). We review ongoing clinical studies investigating the safety and efficacy of PARP inhibitors in the treatment of AML, focusing on specific molecular and genetic AML subgroups that could be particularly sensitive to PARP inhibitor treatment. We also discuss reports describing an increased risk of treatment-related myeloid neoplasms in patients receiving PARP inhibitors for solid tumors. Abstract Despite recent discoveries and therapeutic advances in aggressive myeloid neoplasms, there remains a pressing need for improved therapies. For instance, in acute myeloid leukemia (AML), while most patients achieve a complete remission with conventional chemotherapy or the combination of a hypomethylating agent and venetoclax, de novo or acquired drug resistance often presents an insurmountable challenge, especially in older patients. Poly(ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2, are involved in detecting DNA damage and repairing it through multiple pathways, including base excision repair, single-strand break repair, and double-strand break repair. In the context of AML, PARP inhibitors (PARPi) could potentially exploit the frequently dysfunctional DNA repair pathways that, similar to deficiencies in homologous recombination in BRCA-mutant disease, set the stage for cell killing. PARPi appear to be especially effective in AML with certain gene rearrangements and molecular characteristics (RUNX1-RUNX1T1 and PML-RARA fusions, FLT3- and IDH1-mutated). In addition, PARPi can enhance the efficacy of other agents, particularly alkylating agents, TOP1 poisons, and hypomethylating agents, that induce lesions ordinarily repaired via PARP1-dependent mechanisms. Conversely, emerging reports suggest that long-term treatment with PARPi for solid tumors is associated with an increased incidence of myelodysplastic syndrome (MDS) and AML. Here, we (i) review the pre-clinical and clinical data on the role of PARPi, specifically olaparib, talazoparib, and veliparib, in aggressive myeloid neoplasms and (ii) discuss the reported risk of MDS/AML with PARPi, especially as the indications for PARPi use expand to include patients with potentially curable cancer.

OBJECTIVE: In the Phase 3 VELIA trial (NCT02470585), PARP inhibitor (PARPi) veliparib was combined with first-line chemotherapy and continued as maintenance for patients with ovarian carcinoma enrolled regardless of chemotherapy response or biomarker status. Here, we report exploratory analyses of the impact of homologous recombination deficient (HRD) or proficient (HRP) status on progression-free survival (PFS) and objective response rates during chemotherapy.METHODS: Women with Stage III-IV ovarian carcinoma were randomized to veliparib-throughout, veliparib-combination-only, or placebo. Stratification factors included timing of surgery and germline BRCA mutation status. HRD status was dichotomized at genomic instability score 33. During combination therapy, CA-125 levels were measured at baseline and each cycle; radiographic responses were assessed every 9 weeks.RESULTS: Of 1140 patients randomized, 742 had BRCA wild type (BRCAwt) tumors (HRP, n = 373; HRD/BRCAwt, n = 329). PFS hazard ratios between veliparib-throughout versus control were similar in both BRCAwt populations (HRD/BRCAwt: 22.9 vs 19.8 months; hazard ratio 0.76; 95% confidence interval [CI] 0.53-1.09; HRP: 15.0 vs 11.5 months; hazard ratio 0.765; 95% CI 0.56-1.04). By Cycle 3, the proportion with ≥90% CA-125 reduction from baseline was higher in those receiving veliparib (pooled arms) versus control (34% vs 23%; P = 0.0004); particularly in BRCAwt and HRP subgroups. Complete response rates among patients with measurable disease after surgery were 24% with veliparib (pooled arms) and 18% with control.CONCLUSIONS: These results potentially broaden opportunities for PARPi utilization among patients who would not qualify for frontline PARPi maintenance based on other trials.

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.

Objectives: Previous phase 3 trials of front-line maintenance with the PARP inhibitors (PARPi) olaparib and niraparib only included patients (pts) with complete (CR) or partial (PR) response following front-line chemotherapy (CT); both agents were adopted as standard of care (SOC) in these pts. This prevented the systematic study of all pts who are eligible for PARPi maintenance therapy after front-line CT or to predict their responses. The phase 3 VELIA study (NCT02470585) added veliparib (VEL) to front-line CT at diagnosis, followed by VEL maintenance in the absence of progressive disease (PD), permitting treatment of a broader cohort of pts. This exploratory analysis evaluated baseline characteristics associated with first-line CT response, to understand if predictive variables exist that could identify those who would be eligible vs ineligible for SOC PARPi maintenance. Methods: Pts with untreated Stage III/IV ovarian cancer were randomized to carboplatin/paclitaxel (CP) + placebo (PBO) followed by PBO maintenance (control arm), CP + VEL followed by PBO maintenance (VEL combination only), or CP + VEL followed by VEL maintenance (VEL throughout). Pts without progression after combination treatment transitioned to assigned maintenance therapy. In this exploratory analysis, pts with germline or somatic BRCA mutations (BRCAm) were grouped as maintenance eligible (ME; with CR/PR or non-PD with surgical complete resection) or maintenance not eligible (MNE; with stable disease/progression/non-CR/non-PD). Progression-free survival (PFS) in pts starting maintenance was measured from initiation of maintenance treatment. Results: Of 1,140 randomized pts, 298 had germline or somatic BRCAm; of these, 278 could be classified according to maintenance eligibility. MNE pts (n=56) were distributed equally among the control, VEL combination only, and VEL throughout arms (21%, 19%, and 21% of evaluable pts, respectively). MNE subgroups were enriched for advanced disease (18-39% of MNE subgroups vs 13-19% of ME subgroups had Stage IV disease) and less favorable performance status (41-67% MNE vs 20-40% of ME pts had ECOG performance status ≥1). For ME pts (n=222), median PFS from initiation of maintenance was not reached in the VEL throughout arm vs 19.7 months in the control arm (HR 0.29, 95% CI 0.16-0.52, Figure). For MNE pts, median PFS was 16.4 months in the VEL throughout arm and 18.9 months in the control arm (HR 1.331, 95% CI 0.52-3.44); however, sample sizes (n=18 and n=16, respectively) limit a meaningful analysis of PFS in these pts. Download : Download high-res image (79KB) Download : Download full-size image Conclusions: The results of this exploratory analysis suggest that VEL has robust efficacy in pts with BRCA-mutated tumors eligible for SOC PARPi maintenance. Baseline characteristics of advanced disease and less favorable performance status were more common in MNE pts than ME pts. Molecular characterization is ongoing to identify mechanisms of sensitivity that predict eligibility for, and response to, PARPi therapy.

Objective Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. Methods We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117–166). Results Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). Conclusions Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

OBJECTIVE. This study is designed to identify genes and pathways that could promote metastasis to the bowel in high-grade serous ovarian cancer (OC) and evaluate their associations with clinical outcomes. METHODS. We performed RNA sequencing of OC primary tumors (PTs) and their corresponding bowel metastases (n = 21 discovery set; n = 18 replication set). Differentially expressed genes (DEGs) were those expressed at least 2-fold higher in bowel metastases (BMets) than PTs in at least 30% of patients (P < .05) with no increased expression in paired benign bowel tissue and were validated with quantitative reverse transcription PCR. Using an independent OC cohort (n = 333), associations between DEGs in PTs and surgical and clinical outcomes were performed. Immunohistochemistry and mouse xenograft studies were performed to confirm the role of LRRC15 in promoting metastasis. RESULTS. Among 27 DEGs in the discovery set, 21 were confirmed in the replication set: SFRP2, Col11A1, LRRC15, ADAM12, ADAMTS12, MFAP5, LUM, PLPP4, FAP, POSTN, GRP, MMP11, MMP13, C1QTNF3, EPYC, DIO2, KCNA1, NETO1, NTM, MYH13, and PVALB. Higher expression of more than half of the genes in the PT was associated with an increased requirement for bowel resection at primary surgery and an inability to achieve complete cytoreduction. Increased expression of LRRC15 in BMets was confirmed by immunohistochemistry and knockdown of LRRC15 significantly inhibited tumor progression in mice. CONCLUSIONS. We identified 21 genes that are overexpressed in bowel metastases among patients with OC. Our findings will help select potential molecular targets for the prevention and treatment of malignant bowel obstruction in OC.

Summary Background Based on preclinical work, we found that combination of poly (ADP-ribose) polymerase (PARP) inhibitors with drugs that inhibit the homologous recombination repair (HRR) pathway (such as PI3K inhibitors) might sensitise HRR-proficient epithelial ovarian cancers to PARP inhibitors. We aimed to assess the safety and identify the recommended phase 2 dose of the PARP inhibitor olaparib in combination with the PI3K inhibitor alpelisib in patients with epithelial ovarian cancer and in patients with breast cancer. Methods In this multicentre, open-label, phase 1b trial following a 3 + 3 dose-escalation design, we recruited patients aged 18 years or older with the following key eligibility criteria: confirmed diagnosis of either recurrent ovarian, fallopian tube, or primary peritoneal cancer of high-grade serous histology; confirmed diagnosis of either recurrent ovarian, fallopian tube, or primary peritoneal cancer of any histology with known germline BRCA mutations; confirmed diagnosis of recurrent breast cancer of triple-negative histology; or confirmed diagnosis of recurrent breast cancer of any histology with known germline BRCA mutations. Additional patients with epithelial ovarian cancer were enrolled in a dose-expansion cohort. Four dose levels were planned: the starting dose level of alpelisib 250 mg once a day plus olaparib 100 mg twice a day (dose level 0); alpelisib 250 mg once a day plus olaparib 200 mg twice a day (dose level 1); alpelisib 300 mg once a day plus olaparib 200 mg twice a day (dose level 2); and alpelisib 200 mg once a day plus olaparib 200 mg twice a day (dose level 3). Both drugs were administered orally, in tablet formulation. The primary objective was to identify the maximum tolerated dose and the recommended phase 2 dose of the combination of alpelisib and olaparib for patients with epithelial ovarian cancer and patients with breast cancer. Analyses included all patients who received at least one dose of the study drugs. The trial is active, but closed to enrolment; follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov , number NCT01623349 . Findings Between Oct 3, 2014, and Dec 21, 2016, we enrolled 34 patients (28 in the dose-escalation cohort and six in the dose-expansion cohort); two in the dose-escalation cohort were ineligible at the day of scheduled study initiation. Maximum tolerated dose and recommended phase 2 dose were identified as alpelisib 200 mg once a day plus olaparib 200 mg twice a day (dose level 3). Considering all dose levels, the most common treatment-related grade 3–4 adverse events were hyperglycaemia (five [16%] of 32 patients), nausea (three [9%]), and increased alanine aminotransferase concentrations (three [9%]). No treatment-related deaths occurred. Dose-limiting toxic effects included hyperglycaemia and fever with decreased neutrophil count. Of the 28 patients with epithelial ovarian cancer, ten (36%) achieved a partial response and 14 (50%) had stable disease according to Response Evaluation Criteria in Solid Tumors 1.1. Interpretation Combining alpelisib and olaparib is feasible with no unexpected toxic effects. The observed activity provides preliminary clinical evidence of synergism between olaparib and alpelisib, particularly in epithelial ovarian cancer, and warrants further investigation. Funding Ovarian Cancer Dream Team (Stand Up To Cancer, Ovarian Cancer Research Alliance, National Ovarian Cancer Coalition), Breast Cancer Research Foundation, Novartis.

CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Together, single-cell technologies and systems biology have been used to investigate previously unanswerable questions in biomedicine with unparalleled detail. Despite these advances, gaps in analytical capacity remain. Machine learning, which has revolutionized biomedical imaging analysis, drug discovery, and systems biology, is an ideal strategy to fill these gaps in single-cell studies. Machine learning additionally has proven to be remarkably synergistic with single-cell data because it remedies unique challenges while capitalizing on the positive aspects of single-cell data. In this review, we describe how systems-biology algorithms have layered machine learning with biological components to provide systems level analyses of single-cell omics data, thus elucidating complex biological mechanisms. Accordingly, we highlight the trifecta of single-cell, systems-biology, and machine-learning approaches and illustrate how this trifecta can significantly contribute to five key areas of scientific research: cell trajectory and identity, individualized medicine, pharmacology, spatial omics, and multi-omics. Given its success to date, the systems-biology, single-cell omics, and machine-learning trifecta has proven to be a potent combination that will further advance biomedical research.

ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D mutations and high-level BRCA1 promoter methylation predict response to rucaparib, similar to BRCA1/BRCA2 mutations. BRCA1 methylation loss may be a major cross-resistance mechanism to platinum and PARPi. Genomic scars associated with homologous recombination deficiency are irreversible, persisting even as platinum resistance develops, and therefore are predictive of rucaparib response only in platinum-sensitive disease. The RAS, AKT, and cell cycle pathways may be additional modulators of PARPi sensitivity., The identification of biomarkers of response to PARP inhibitors can enable selection of appropriate ovarian cancer patients for treatment. In this study, the authors report clinical results and exploratory biomarker analyses from the ARIEL2 phase 2 clinical trial on the safety and efficacy of the PARP inhibitor rucaparib in patients with recurrent ovarian cancers

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting., The authors would like to thank Dr. Kenneth McCreath for editorial support. This work was supported by the NIH National Cancer Institute Grants R01 CA116623 (to Ruth Lupu) and R01 CA166741 (to Scott H. Kaufmann) and by the U.S. Department of Defense (DOD)-Breakthrough 3 Grants BC151072 and BC151072P1 (to Ruth Lupu). Work in the Menendez laboratory is supported by the Spanish Ministry of Science and Innovation (Grants SAF2016-80639-P and PID2019-10455GB-I00, Plan Nacional de l + D + I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona). Joan Montero acknowledges support from the Ramon y Cajal Programme, Ministerio de Economía y Competitividad (RYC-2015-18357) and the Spanish National Plan “Retos Investigación” I + D + I (RTI2018-094533-A-I00) from the Ministerio de Ciencia, Innovación y Universidades. Elisabet Cuyàs holds a research contract “Miguel Servet” (CP20/00003) from the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (Spain). All authors have read and agreed to the published version of the manuscript.

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. Significance: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine–rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody–drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases. Significance: This study identifies that LRRC15 activates β1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody–drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism, and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML), and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy–lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB–IDH1/IDH2–TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by an MYC–TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML. Significance: Alterations in epigenetic control are a hallmark of AML. This study establishes that a MYC–TFEB circuit controls AML differentiation and epigenetic programs by inducing IDH1/IDH2 and hydroxylation of 5-methylcytosine, that TFEB functions as a tumor suppressor in AML, and that this circuit is a targetable vulnerability in AML. See related commentary by Wu and Eisenman, p. 116.

Long-term treatment outcomes for patients with high grade ovarian cancers have not changed despite innovations in therapies. There is no recommended assay for predicting patient response to second-line therapy, thus clinicians must make treatment decisions based on each individual patient. Patient-derived xenograft (PDX) tumors have been shown to predict drug sensitivity in ovarian cancer patients, but the time frame for intraperitoneal (IP) tumor generation, expansion, and drug screening is beyond that for tumor recurrence and platinum resistance to occur, thus results do not have clinical utility. We describe a drug sensitivity screening assay using a drug delivery microdevice implanted for 24 h in subcutaneous (SQ) ovarian PDX tumors to predict treatment outcomes in matched IP PDX tumors in a clinically relevant time frame. The SQ tumor response to local microdose drug exposure was found to be predictive of the growth of matched IP tumors after multi-week systemic therapy using significantly fewer animals (10 SQ vs 206 IP). Multiplexed immunofluorescence image analysis of phenotypic tumor response combined with a machine learning classifier could predict IP treatment outcomes against three second-line cytotoxic therapies with an average AUC of 0.91.

Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian 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 1832.

Background: Large scale genetic perturbations screens have been instrumental for many key discoveries. In the last decade sequence specific genetic perturbations created RNAi and CRISPR (PMID: 25446316). Recently, specific genome targeting approaches using single guide RNAs (sgRNAs) for the CRISPR-Cas9 system have been repurposed to induce sequence-specific repression or activation of gene expression at a genome-wide scale. Different perturbation approaches have different effects and encompass a trade-off between specificity and duration in establishing a measurable perturbation which can result in different biological readouts. However, bench-side strategies are time consuming and expensive. The method described here aims to build a systems-based computational platform to assess pharmacological and biological perturbations at genome-wide network level.Methods: Our network tool NetDecoder (Nucleic Acids Res. 2016; 44(10):e100) that models genome-wide information flow will be used to build context specific protein-protein interaction networks. These networks will be subsequently subjected to in silico perturbations and their impacts on network properties will be assessed. AVANA and Demeter datasets will be used during method validation procedures. Results: Using publicly available expression datasets we generated context specific Breast Cancer networks and individually manipulated nodes and edges of context-specific networks associated with pharmacological FDA approved targets. Our perturbation networks captured cellular rewiring by engulfing new gene players, alterations of the network size and its connectivity. Conclusions: Our in-silico genome wide perturbation platform provides a flexible approach to fine-tune single and tandem manipulations, allowing for inexpensively monitoring a range of pharmacological interventions using large omics data. Citation Format: Cristina Correia, Choong Yong Ung, Cheng Zhang, Scott H. Kaufmann, Hu Li. A genome wide pharmacological perturbation platform [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 1226.

IMPORTANCE: A total of 1% to 3% of patients treated with a poly(adenosine diphosphate–ribose) polymerase inhibitor for high-grade ovarian cancer (HGOC) develop therapy-related myeloid neoplasms (t-MNs), which are rare but often fatal conditions. Although the cause of these t-MNs is unknown, clonal hematopoiesis of indeterminate potential (CHIP) variants can increase the risk of primary myeloid malignant neoplasms and are more frequent among patients with solid tumors. OBJECTIVES: To examine whether preexisting CHIP variants are associated with the development of t-MNs after rucaparib treatment and how these CHIP variants are affected by treatment. DESIGN, SETTING, AND PARTICIPANTS: This retrospective genetic association study used peripheral blood cell (PBC) samples collected before rucaparib treatment from patients in the multicenter, single-arm ARIEL2 (Study of Rucaparib in Patients With Platinum-Sensitive, Relapsed, High-Grade Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer) (n = 491; between October 30, 2013, and August 9, 2016) and the multicenter, placebo-controlled, double-blind ARIEL3 (Study of Rucaparib as Switch Maintenance Following Platinum-Based Chemotherapy in Patients With Platinum-Sensitive, High-Grade Serous or Endometrioid Epithelial Ovarian, Primary Peritoneal or Fallopian Tube Cancer) (n = 561; between April 7, 2014, and July 19, 2016), which tested rucaparib as HGOC therapy in the treatment and maintenance settings, respectively. The follow-up data cutoff date was September 1, 2019. Of 1052 patients in ARIEL2 and ARIEL3, PBC samples from 20 patients who developed t-MNs (cases) and 44 randomly selected patients who did not (controls) were analyzed for the presence of CHIP variants using targeted next-generation sequencing. Additional longitudinal analysis was performed on available ARIEL2 samples collected during treatment and at the end of treatment. MAIN OUTCOMES AND MEASURES: Enrichment analysis of preexisting variants in 10 predefined CHIP-associated genes in cases relative to controls; association with clinical correlates. RESULTS: Among 1052 patients (mean [SE] age, 61.7 [0.3] years) enrolled and dosed in ARIEL2 and ARIEL3, 22 (2.1%) developed t-MNs. The t-MNs were associated with longer overall exposure to prior platinum therapies (13.2 vs 9.0 months in ARIEL2, P = .04; 12.4 vs 9.6 months in ARIEL3, P = .003). The presence of homologous recombination repair gene variants in the tumor, either germline or somatic, was associated with increased prevalence of t-MNs (15 [4.1%] of 369 patients with HGOC associated with an HRR gene variant vs 7 [1.0%] of 683 patients with wild-type HGOC, P = .002). The prevalence of preexisting CHIP variants in TP53 but not other CHIP-associated genes at a variant allele frequency of 1% or greater was significantly higher in PBCs from cases vs controls (9 [45.0%] of 20 cases vs 6 [13.6%] of 44 controls, P = .009). TP53 CHIP was associated with longer prior exposure to platinum (mean 14.0 months of 15 TP53 CHIP cases vs 11.1 months of 49 non-TP53 CHIP cases; P = .02). Longitudinal analysis showed that preexisting TP53 CHIP variants expanded in patients who developed t-MNs. CONCLUSIONS AND RELEVANCE: The findings of this genetic association study suggest that preexisting TP53 CHIP variants may be associated with t-MNs after rucaparib treatment.

Although the C H chains of petroleum derivatives display unique absorption features in the short-wave infrared (SWIR), it is a challenge to identify plastics on terrestrial surfaces. The diverse reflectance spectra caused by chemically varying polymer types and their different kinds of brightness and transparencies, which are, moreover, influenced further by the respective surface backgrounds. This paper investigates the capability of WorldView-3 (WV-3) satellite data, characterized by a high spatial resolution and equipped with eight distinct and relatively narrow SWIR bands suitable for global monitoring of different types of plastic materials. To meet the objective, hyperspectral measurements and simulations were conducted in the laboratory and by aircraft campaigns, based on the JPL-ECOSTRESS, USGS, and inhouse hyperspectral libraries, all of which are convolved to the spectral response functions of the WV-3 system. Experiments further supported the analyses wherein different plastic materials were placed on different backgrounds, and scaled percentages of plastics per pixel were modeled to determine the minimum detectable fractions. To determine the detectability of plastics with various chemical and physical properties and different fractions against diverse backgrounds, a knowledge-based classifier was developed, the routines of which are based on diagnostic spectral features in the SWIR range. The classifier shows outstanding results on various background scenarios for lab experimental imagery as well as for airborne data and it is further able to mask non-plastic materials. Three clusters of plastic materials can clearly be identified, based on spectra and imagery: The first cluster identifies aliphatic compounds, comprising polyethylene (PE), polyvinylchloride (PVC), ethylene vinyl acetate copolymer (EVAC), polypropylene (PP), polyoxymethylene (POM), polymethyl methacrylate (PMMA), and polyamide (PA). The second and third clusters are diagnostic for aromatic hydrocarbons, including polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), and styrene-acrylonitrile (SAN), respectively separated from polybutylene adipate terephthalate (PBAT), acrylonitrile butadiene styrene (ABS), and polyurethane (PU). The robustness of the classifier is examined on the basis of simulated spectra derived from our HySimCaR model, which has been developed in-house. The model simulates radiation transfer by using virtual 3D scenarios and ray tracing, hence, enables the analysis of the influence of various factors, such as material brightness, transparency, and fractional coverage as well as different background materials. We validated our results by laboratory and simulated datasets and by tests using airborne data recorded at four distinct sites with different surface characteristics. The results of the classifier were further compared to results produced by another signature-based method, the spectral angle mapper (SAM) and a commonly used technique, the maximum likelihood estimation (MLE). Finally, we applied and successfully tested the classifier on WV-3 imagery of sites known for a high abundance of plastics in Almeria (Spain), Cairo (Egypt), and Accra, (Ghana, West Africa). Both airborne and WV-3 data were atmospherically corrected and transferred to “at-surface reflectances”. The results prove the combination of WV-3 data and the newly designed classifier to be an efficient and reliable approach to globally monitor and identify three clusters of plastic materials at various fractions on different backgrounds.