bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B051: PARG inhibition leads to the formation of toxic nuclear PARP1 aggregates
Authors: Paradkar Sateja , Cui Annie , Purcell Julia , Friedman Sam , Jensen Ryan , Bindra Ranjit
Published: December 2023

Mutations in DNA repair genes, such as BRCA1/2 mutations, occur in 50-70% of breast cancers among others. PARP inhibitors (PARPis) have shown remarkable efficacy against these tumors, however, emerging clinical evidence has revealed that PARPi resistance represents a major concern in these cancers with up to 90% of initial responders eventually developing resistance to the therapy. To overcome the limitations of PARPis, researchers have been working towards developing PARG inhibitors (PARGis) for the treatment of DNA repair-deficient cancers.   In response to DNA damage, PARP1 synthesizes poly(ADP-ribose) (PAR) chains at break sites, which are crucial for the proper release of PARP1 and the recruitment of downstream repair proteins to the break site. PAR glycohydrolase (PARG) is the primary enzyme responsible for the resolution of PAR chains following repair. Owing to their central role in PARP-dependent processes, PARG inhibitors are currently being developed for cancer therapy, however, their precise mechanism of action has yet to be determined. Here we report that PARG loss or inhibition leads to cytotoxic effects through the formation of toxic PARP1 aggregates in response to DNA damage. Using live cell imaging assays, we determined that PARP1 aggregates form at sites distant from the original site of damage, causing a mislocalization of DNA repair factors from the damage site. We also find that these aggregates are formed by and composed of long, branched PAR chains, which are likely sequestering ATP and NAD+ reserves in the nucleus. Finally, we show that these aggregates are associated with increased cell death in BRCA-deficient cell lines suggesting that these aggregates are critical for PARGi mechanism of toxicity. Overall, this work elucidates the mechanism of action of PARG inhibitors, which will help identify patient populations in which these drugs could be used.

Citation Format: Sateja Paradkar, Annie Cui, Julia Purcell, Sam Friedman, Ryan Jensen, Ranjit Bindra. PARG inhibition leads to the formation of toxic nuclear PARP1 aggregates [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B051.
Abstract B053: Applying a new drug discovery platform for the discovery and development of inhibitors of DDR proteins APE1 and Pol eta
Authors: Das Debanu , Duncton Matthew , Deacon Ashley , III David Wilson
Published: December 2023

Quickly generating reliable hits from library screening and rapidly converting them into lead compounds, which can be verified in functional assays, is critical in early preclinical drug discovery. The expedited validation of “druggability” of targets and identification of modulators to advance to preclinical studies can significantly increase the success of drug discovery and development. Our SaXPy (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows (i) hit generation; (ii) mapping of protein-ligand interactions; (iii) assessment of target ligandability; (iv) discovery of novel and potential allosteric binding sites; and (v) hit-to-lead progression. These advances inform chemical tractability, downstream biology, and intellectual property. We describe here the application of SaXPy in the discovery and development of DNA Damage Response (DDR) inhibitors against DNA polymerase eta (Pol 𝝶 or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our studies resulted in the first crystal structures of these targets bound to small molecules and the discovery of novel, multiple binding sites, which we could quickly progress from hit-to-lead development and demonstrate functional activity in bioassays.

Citation Format: Debanu Das, Matthew Duncton, Ashley Deacon, David Wilson III. Applying a new drug discovery platform for the discovery and development of inhibitors of DDR proteins APE1 and Pol eta [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B053.
Abstract B056: Modulation of the DNA replication stress response and chemo-response by the mono-ADP-ribosyltransferase PARP14
Authors: Dhoonmoon Ashna , Nicolae Claudia M , Moldovan George-Lucian
Published: December 2023

Genomic instability is a major hallmark of cancer and one of the most common causes is DNA replication stress. Mutations in genes involved in the DNA replication stress response are closely linked to cancer predisposition and progression but this has been exploited clinically. In the advent of personalized medicine, it has become clear that a better understanding of underlying mechanisms is important to advance cancer therapy, especially to address chemoresistance. Here, we identified novel roles for PARP14, a lesser studied member of the PARP family, in regulation of the replication stress response, and subsequently genomic stability and chemo-response. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we first catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Furthermore, we showed that PARP14 plays a central role in stabilization of stalled replication forks which arise due to high replication stress level in BRCA1/2 deficient cells. Mechanistically, we showed that PARP14 mediates stalled replication fork degradation via its interaction with the nuclease MRE11. Moreover, we also identified KU as an essential component of a multi-step mechanism by which the KU-PARP14 axis differentially promotes degradation by the nucleases MRE11 and EXO1. This mechanism restores genomic stability in BRCA-deficient cells and subsequently promotes chemoresistance. Overall, our work provides insights into novel mechanisms by which PARP14 affects the replication stress response and demonstrates that PARP14 can be used as a biomarker for chemo-response.

Citation Format: Ashna Dhoonmoon, Claudia M Nicolae, George-Lucian Moldovan. Modulation of the DNA replication stress response and chemo-response by the mono-ADP-ribosyltransferase PARP14 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B056.
Abstract B064: Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells
Authors: Vitor Alexandra , Benfatto Salvatore , Zenke Frank , Waszak Sebastian , Mardin Balca
Published: December 2023

Mismatch repair (MMR) deficiency is commonly found in tumors with high mutational burden. Although the mutational signatures of these deficiencies are determined, the initial trajectories these cells follow to reach a tumorigenic state with high mutational load have not been studied due to lack of isogenic model systems. For this reason, we generated three isogenic MMR deficient cell lines from a normally untransformed cell line with few genomic abnormalities and low mutational burden. After continued culturing of these MMR-deficient cell lines for several months, we profiled them by single cell RNA sequencing using the 10X Chromium platform. Next, we identified different clusters of cells in MMR deficient cell lines that acquire tumorigenic properties. In these clusters, we identified gene expression signatures that are responsible from the tumorigenic properties of these clusters of cells. After isolation of the single clones that bear these gene expression signatures, we characterized how MMR deficiency can give rise to tumors with particular gene expression trajectories. In addition, we performed CRISPR/Cas9-based genome-wide screens to understand the vulnerabilities of these clusters of cells.  Altogether, our results shed light on the early steps of tumorigenesis in MMR deficiency.

Citation Format: Alexandra Vitor, Salvatore Benfatto, Frank Zenke, Sebastian Waszak, Balca Mardin. Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B064.
Abstract B069: Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors
Authors: Noronha Katelyn , Liu Samantha , Lucas Karlie , Bindra Ranjit S.
Published: December 2023

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in approximately 80% of secondary glioblastomas and low-grade gliomas and to a lesser extent in AML, cholangiocarcinoma, colorectal cancer, and chondrosarcomas. Mutations in IDH1/2 lead to the overproduction of 2-hydroxyglutarate (2HG), which has been named an “oncometabolite” and is implicated in cancer progression. Work by our group has shown that 2HG induced hypermethylation suppresses homologous recombination repair. As a result, cells with mutations in IDH1/2 accumulate double strand breaks (DSBs) that must be addressed by other modes of DSB repair, such as canonical NHEJ or polymerase theta mediated end joining (TMEJ). Furthermore, TMEJ is understudied in IDH1/2-mutant cancers but has been studied extensively in other HR-deficient cancer contexts, such as BRCA-mutant cancers. Conflicting reports remain on whether TMEJ promotes genomic stability or instability in these contexts, due to its error prone repair. To better understand how IDH1/2-mutant cells utilize TMEJ and polymerase theta’s impact on genomic stability, we studied the effects of polymerase theta inhibitors on isogenic cell line models of the IDH1R132H mutation. We find that mRNA levels of polymerase theta are increased in IDH1-mutant cell lines. Using a polymerase theta inhibitor, ART558, we also observed reduced viability in IDH1-mutant cells compared to parental cells. This loss of viability in IDH1-mutant cells is likely a result of the observed increase of gamma H2AX subnuclear foci in IDH1-mutant cells treated with ART558, which is a marker of unresolved DNA damage. Preliminary data suggests that this unrepaired damage is contributing to an overall increase in micronuclei formation in IDH1-mutant cells treated with ART558 as well. Taken together, these results suggest that IDH1-mutant cells upregulate polymerase theta expression to promote genomic stability through repair of persistent DSBs. This study on TMEJ in IDH1/2 mutant cancers has identified a therapeutic vulnerability in these HR-deficient cancers that can be targeted with polymerase theta inhibitors.

Citation Format: Katelyn Noronha, Samantha Liu, Karlie Lucas, Ranjit S. Bindra. Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B069.
Abstract B070: Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells
Authors: Ham Sujin , Kim So Hyeon , Hwang Hae Min , Lee Minyoung , Noh Youlim , Lee Changyun , Kim Yu-Jin , Kim Jinyong , Lee Dae-Won , Lee Kyung-Hun , Im Seock-Ah
Published: December 2023

Background: Poly (ADP-ribose) polymerase (PARP) 1 has multiple cellular functions including its crucial role in DNA damage response. PARP inhibitor (PARPi) blocks the synthesis of PAR polymers and traps PARP1 on the damage sites. Currently approved PARPis target both PARP1 and PARP2, but the inhibition of PARP2 can cause undesirable hematological toxicity. AZD5305 is a novel selective PARP1 inhibitor that can reduce the hematological toxicity by off-targeting of PARP2. We evaluated the anti-tumor effects and mechanism of action of AZD5305 in breast and gastric cancer cells in vitro.

Methods: To investigate the anti-tumor effects of AZD5305, we used 2 cell lines of breast cancer cells (HCC1395 and MCF7) and 3 cell lines of human gastric cancer cells (SNU-601, SNU-668 and KATO Ⅲ). The cytotoxicity of AZD5305 was evaluated by colony formation assay (CFA) for 14 days with increasing concentration of AZD5305 (doses range: 0-5nM). Cell cycle was analyzed by flow cytometry and apoptotic cell death was assessed by annexin-Ⅴ/PI staining. DNA damage was detected by comet assay and cell immunofluorescence assay.

Results: BRCA1/2 mutated HCC1395 and RAD51C deficient SNU-601 were sensitive to AZD5305 (IC50: 0.25 nM), and SNU-668 was moderately sensitive (IC50: 2.05nM), while MCF7 and KATO III were less-sensitive to AZD5305 (IC50 > 5 nM). AZD5305 induced the G2/M cell cycle arrest in a dose-dependent manner in HCC1395 and SNU-601, but not in MCF7 and KATO Ⅲ. Moreover, AZD5305 increased sub-G1 population in HCC1395 and SNU-601 cells indicating apoptotic cell death, which was also confirmed by annexin-V assay and the cleaved PARP and caspase-7. Phosphorylation of RPA, γH2AX, and the expression of molecules related to DNA damage response were increased in the sensitive cells. Moreover, comet tails which indicate fragmented DNA were observed in the sensitive cells, but not in insensitive cells.

Conclusions: AZD5305, a novel PARP1 selective inhibitor, shows cytotoxic effect in HR defective breast and gastric cancer cells in vitro. AZD5305 accumulates DNA damage which leads to apoptotic cell death in sensitive cell lines, but not in less-sensitive cell lines.

Citation Format: Sujin Ham, So Hyeon Kim, Hae Min Hwang, Minyoung Lee, Youlim Noh, Changyun Lee, Yu-Jin Kim, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B070.
Abstract B091: Overexpression of Muscleblind Like Splicing Regulator 2 (MBNL2) enhances cisplatin resistance in ovarian cancer
Authors: Ju Woong , Sung Hye Youn , Ahn Jung-Hyuck
Published: December 2023

Ovarian cancer is the second most commonly diagnosed gynecological cancer and has the highest mortality rate of all gynecological cancers worldwide. Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin is a major barrier to successful therapy. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified Muscleblind Like Splicing Regulator 2 (MBNL2) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, MBNL2 mRNA expression was significantly upregulated, and the MBNL2 promoter was associated with hypomethylation of the specific cytosine-phosphate-guanine (CpG) sites. Reduced MBNL2 expression in cisplatin-sensitive cell lines was restored by treatment with a DNA demethylation agent, suggesting epigenetic regulation of MBNL2 expression by promoter methylation. Furthermore, depletion of MBNL2 in cisplatin-resistant cells induced cytotoxicity in response to cisplatin, whereas overexpression of MBNL2 in cisplatin-sensitive cells increased chemoresistance, indicating that MBNL2 plays a vital role in developing chemoresistance in ovarian cancer cells. Furthermore, we performed meta-analysis using fourteen publicly available datasets to assess the association between expression of MBNL2 and overall survival in patients with serous-type ovarian cancer. The pooled Hazard ratios (HRs) estimate was significantly greater than 1 for overall survival (HR=1.09, p-value=0.0034). The association was more significant (HR=1.12, p-value=0.001) when tumor stage was added to the survival analysis as a covariate. The meta-analysis revealed that high expression of MBNL2 was significantly associated with poor prognosis and a higher risk of death in ovarian cancer patients with serous tumor. Additionally, we further validated the MBNL2 mRNA expression in primary ovarian tumor tissues. The statistically significant upregulation of MBNL2 mRNA expression was observed in cisplatin-resistant patients compared to that of cisplatin-sensitive patients. Our findings suggest MBNL2 may be a potential therapeutic target for the prevention of chemoresistance to cisplatin in ovarian cancer.

Citation Format: Woong Ju, Hye Youn Sung, Jung-Hyuck Ahn. Overexpression of Muscleblind Like Splicing Regulator 2 (MBNL2) enhances cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B091.
Abstract B094: Pentraxin 3 promotes the therapy resistance to sorafenib via regulating apoptosis in hepatocellular carcinoma
Authors: Chen Ping-Wen
Published: December 2023

Hepatocellular carcinoma (HCC) is prevalently regarded as an inflammation-related cancer, characterized by persistent inflammation throughout the tumor microenvironment (TME). Currently, Sorafenib is the first-line targeted therapy drug used for advanced HCC patients. Unfortunately, many patients still face the risk of drug resistance after treatment with sorafenib. Pentraxin 3 (PTX3) has been reported as a secretory protein involved in cancer-related inflammation. It has been found to be overexpressed and acts as an unfavorable prognostic factor in the progression of chronic inflammation-related cancers. A recent study indicated that HCC patients who received sorafenib treatment had significantly higher PTX3 expression. However, the detailed role of PTX3 in these sorafenib-treated HCC patients still remains unclear. Therefore, it is intriguing whether elevated PTX3 contributes to sorafenib resistance in HCC patients. In this study, we observed that PTX3 is responsive to sorafenib, and there is a positive correlation between PTX3 expression and sorafenib resistance. Depletion of PTX3 led to increased sensitivity of HCC cells to sorafenib. For the underlying mechanism, sorafenib-induced PTX3 was found to promote resistance by regulating the anti-apoptotic protein MCL-1 and the pro-apoptotic protein BAX. In vivo experiments also revealed that combined treatment of sorafenib and a PTX3 inhibitor significantly reduced the size of HCC tumors, demonstrating that blocking elevated PTX3 increases sensitivity to sorafenib. Overall, targeting PTX3 attenuates resistance to sorafenib through the regulation of apoptosis, providing a novel therapeutic strategy to enhance therapeutic efficacy in HCC patients undergoing sorafenib treatment.

Citation Format: Ping-Wen Chen. Pentraxin 3 promotes the therapy resistance to sorafenib via regulating apoptosis in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B094.
Abstract B115: Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor
Authors: Iyer Chandrasekar , Li Binghui , Stewart Trent R , Wang Tao , Capen Andrew , Cavitt Rachel , Anderson Bryan D , Bocchinfuso Wayne , Zhao Gaiying , Rodriguez Michael J , Carballares Santiago , Cooke Andrew , Bondi Robert , Burns Lee , Kelamangalath Lakshmi , Wallace Ross , Kolakowski Gabrielle , Henry James R , Si Chong
Published: December 2023

KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively and less commonly in other cancers. Here, we describe LY3962673, a highly potent inhibitor of KRAS G12D that is selective against wild-type (WT) KRAS and orally bioavailable. Compound potency and selectivity were measured using surface plasmon resonance (SPR) and cell-based assays monitoring the inhibition of pERK (Thr202/Tyr204) and cell growth. In SPR assays, LY3962673 had Kd values of 0.058 and 4.7 nM for GDP-bound KRAS G12D and WT KRAS, respectively (81-fold selective). In KRAS G12D-mutant HPAC, LY3962673 inhibited ERK1/2 phosphorylation with an IC50 of 4.2 nM contrasting 231 nM IC50 in WT KRAS MKN45 cells (55-fold selective). Similarly, LY3962673 inhibited cell growth in KRAS G12D and WT KRAS cell lines with IC50 values of 7.7 nM and 1800 nM, respectively (233-fold selective).  LY3962673 shows a clean safety profile in a 133 off-target panel screen. LY3962673 demonstrates favorable in vitro ADME properties. LY3962673 shows oral bioavailability across preclinical species when dosed either as freebase or salt form. Tumor growth inhibition and PK/PD studies were performed in mice. In vivo, LY3962673 administered orally demonstrated tumor regressions in KRAS G12D-driven tumor models, without body weight loss. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients. These data demonstrate that LY3962673 potently inhibits KRAS G12D while sparing WT KRAS and other off-targets with its selectivity. We hypothesize that LY3962673’s low nanomolar potency, desirable selectivity profile, and oral bioavailability will provide efficacy and tolerability in patients with KRAS G12D-driven cancers. An IND submission is planned in the first half of 2024.

Citation Format: Chandrasekar Iyer, Binghui Li, Trent R Stewart, Tao Wang, Andrew Capen, Rachel Cavitt, Bryan D Anderson, Wayne Bocchinfuso, Gaiying Zhao, Michael J Rodriguez, Santiago Carballares, Andrew Cooke, Robert Bondi, Lee Burns, Lakshmi Kelamangalath, Ross Wallace, Gabrielle Kolakowski, James R Henry, Chong Si. Preclinical characterization of LY3962673, an orally bioavailable, highly potent, and selective KRAS G12D inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B115.
Abstract B141: Evaluation of antigen-agnostic anti-tumor activity and immunological memory induced by CBX-15 (alphalexTM-MMAE) in the rat syngeneic breast cancer model
Authors: Gayle Sophia , Paradis Timothy , Zhang Qing , Tylaska Laurie , Hagen Connor , Pasqualini Theresa , Paralkar Vishwas
Published: December 2023

CBX-15 is a peptide-drug conjugate consisting of alphalexTM-monomethyl auristatin E (MMAE). The alphalexTM is a unique variant of pH-Low Insertion Peptide1 (pHLIP®) designed to target the low pH microenvironment of cancer cells, a universal feature of all rapidly growing tumors. The peptide of CBX-15 forms an alpha helix only in low pH conditions, resulting in unidirectional insertion of the peptide and delivery of MMAE across the cancer cell membrane, and avoidance of delivery to healthy tissues, including immune cells.  Efficacy and anti-tumor immunological memory induced by CBX-15 was evaluated in Fischer 344 rats bearing syngeneic 13762 mammary adenocarcinoma tumors. The development of anti-tumor immunological memory was examined in CBX-15-cured animals by in vivo/ex vivo rechallenge with live tumor cells and subsequent assessment of tumor rejection, cytokine release by T cells, tumor immune cell infiltration, and memory T cell composition of the bone marrow.  CBX-15 rapidly regressed rat tumors, resulting in complete responses while sparing healthy tissues such as bone marrow. Cured rats rejected live tumor rechallenge and exhibited a doubling of bone marrow-resident CD4 memory T cells 50 days post-dose. Splenocytes and lymph node suspensions derived from cured rats demonstrated formation of a Th1-mediated IFNƳ response when exposed ex vivo to tumor cells.   The ability of CBX-15 to induce immunogenic cell death was established by vaccinating syngeneic animals with CBX-15 treated tumor cells and subsequent tumor rechallenge, which demonstrated anti-tumor immunity induced by CBX-15.  These preclinical data demonstrate the anti-tumor efficacy of CBX-15 in the rat as well as the ability of CBX-15 to enhance tumor immunogenicity through immunogenic cell death by utilizing a universal pH-based tumor targeting mechanism.1 Wyatt LC et al. 2017. Applications of pHLIP Technology for Cancer Imaging and Therapy. Trends Biotech.

Citation Format: Sophia Gayle, Timothy Paradis, Qing Zhang, Laurie Tylaska, Connor Hagen, Theresa Pasqualini, Vishwas Paralkar. Evaluation of antigen-agnostic anti-tumor activity and immunological memory induced by CBX-15 (alphalexTM-MMAE) in the rat syngeneic breast cancer model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B141.
Abstract B151: Discovery and characterization of a novel small molecule brain penetrant PD-L1 inhibitor
Authors: Zhang Yongxian , Zhang Mingming , Zhao Baowei , Yu Hongping , Xu Yao-Chang , Chen Zhui , Ying Haiyan
Published: December 2023

Introduction: Immunotherapy has revolutionized cancer treatment in the last decade. Several monoclonal PD-1 and PD-L1 antibodies have been approved for treating various cancers. Small molecule PD-L1 inhibitors with brain-penetrating ability may have potential to overcome the limitations of antibodies and bring benefit for patients with intracranial tumors. Leveraging advanced computation-aided structural analysis and medicinal chemistry design, we have successfully discovered an innovative orally available small molecule PD-L1 inhibitor ABSK044. In preclinical experiments, this compound demonstrates robust T-cell activating ability, strong anti-tumor efficacy, and brain-penetrating activity.

Method: ABSK044's ability to block PD-1-PD-L1 interaction was evaluated by HTRF assay and cellular luciferase reporter assay. Its biological activity was evaluated in vitro in MLR and in vivo in syngeneic tumor models.

Results: ABSK044 strongly inhibits PD-1-PD-L1 interaction with an IC50 less than 1nM in vitro and very potently rescues PD-L1-induced suppression of T cell activation signaling in cells. Furthermore, it efficiently rescues cytokine production in CD8+ T cells suppressed by PD-L1, reaching a level comparable to that of PD-L1 antibodies. In in vivo studies, oral administration of ABSK044 strongly inhibits tumor growth to an extent similar to therapeutic anti-PD-L1 antibodies. Notably, ABSK044 demonstrates excellent brain penetration with a Kp value exceeding 0.4. DMPK and safety profiling demonstrate excellent drug-like properties of ABSK044.

Conclusion: ABSK044, presented here by Abbisko Therapeutics, is a highly potent and orally available small molecule PD-L1 antagonist with brain-penetrating activity. Its superior profile supports its fast-track preclinical development.

Citation Format: Yongxian Zhang, Mingming Zhang, Baowei Zhao, Hongping Yu, Yao-Chang Xu, Zhui Chen, Haiyan Ying. Discovery and characterization of a novel small molecule brain penetrant PD-L1 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B151.
Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers
Authors: Chen Yaoyu , Chen Jinhong , Guo Yali , Pang Wei , Zhou Tingru , Liu Xipan , Schomer Nathan , Li Fang , Liang Jiaqi , Sheng Qing
Published: December 2023

Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities.

Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B173.
Abstract C004: Liquid biopsy based on the lipid profiles in plasma-derived extracellular vesicles which can be utilized for breast cancer diagnosis
Authors: Kawashima Masahiro
Published: December 2023

Lipids are a major component of cells that can influence multiple cellular functions. Our group previously found that the breast cancer cells could dramatically change their lipid profile during their progression. Thus, we hypothesized that the lipid profile of extracellular vesicles (EVs) could be used as a biomarker for breast cancer (BC) since the lipid profiles of breast cancer cells can be reflected in that of EVs secreted from cancer cells. In the present study, we optimized a method to isolate EVs from human plasma samples that could be used for the comprehensive lipid analysis. We then explored specific lipid profiles in plasma-derived EVs that could discriminate BC and healthy individuals, tumor subtypes and stages. Plasma-derived EVs from breast cancer (n = 105), benign (n = 11), and healthy individuals (n = 43) were used for lipid analysis. The results showed that the change in proportion of the lipids in the EVs could well discriminate stages and subtypes of BC. The proportion of major phospholipids, phosphatidylethanolamines, phosphatidylserines, phosphatidylcholines (PCs) and phosphatidylinositols (PIs) showed different patterns at different stages. Importantly, PI (18:0/20:3), which we previously identified as a lipid biomarker of disease progression expressed in the primary breast cancer tissue, showed a similar trend in the plasma-derived EVs. This finding supports our initial hypothesis. Besides the phospholipids, sphingomyelins were found to be higher in stage IV. With regard to tumor subtypes, PCs and ceramides were enriched in HER2-positive subtype, while lysoPCs and polyunsaturated triglycerides were enriched in triple negative subtype. Furthermore, a BC diagnostic model consisting of three lipids was successfully established and the model showed good performance in discriminating between healthy individuals and BC in the two independent data sets (the area under the ROC curve was estimated around 0.759 to 0.804). In conclusion, profiling of lipids in plasma-derived EVs has the potential to be used as a liquid biopsy that can diagnose BC, and its stages and subtypes. Further validation and improvement in EV isolation technique would be needed to achieve sufficient performance sufficient for clinical application.

Citation Format: Masahiro Kawashima. Liquid biopsy based on the lipid profiles in plasma-derived extracellular vesicles which can be utilized for breast cancer diagnosis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C004.
Abstract C018: Prognostic worth of Nrf2/BACH1/HO-1 signals in the development of breast cancer
Authors: Barnes Precious , Saahene Roland Osei
Published: December 2023

Nrf2/BACH1/HO-1 signals have been implicated in the development and progression of tumors. However, there has been no comprehensive analysis of their expressions in breast cancer. This study evaluated Nrf2/BACH1/HO-1 signals expression and its clinical significance in breast cancer. 114 female breast cancer of different pathologic parameters and non-cancerous tissues were evaluated for Nrf2/BACH1/HO-1 signals expression using immunohistochemistry and western blot.  The relationship connecting Nrf2/BACH1/HO-1 expression and clinicopathologic factors were assessed using the chi-square test. Nrf2 protein in cancerous tissues (74%) was significantly higher than in the non-cancerous tissues (43% p < 0.002), BACH1 expression in cancerous tissues (61%) was significantly lower than that of non-cancerous tissues (20% p < 0.031) and HO-1 protein expression in cancerous tissues (67%) was highly significant than in the non-cancerous tissues (17% p < 0.001). The expressions of Nrf2 and HO-1 significantly correlated with tumor grade whilst BACH1 was significantly associated with tumor stage (p < 0.05). Nrf2, BACH1 and HO-1 play key roles in the growth and development of breast cancer and can serve as probable biological markers for breast cancer diagnosis, prognosis as well as therapeutic targets.

Citation Format: Precious Barnes, Roland Osei Saahene. Prognostic worth of Nrf2/BACH1/HO-1 signals in the development of breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C018.
Abstract C040: Discovery and development of novel ATG13 degrading compounds that inhibit autophagy and treat Non-Small Cell Lung Cancer
Authors: Hagan Patrick M , Emami Atoosa , Austin Hilarie , Pass Ian , Limpert Allison , Sheffler Douglas , Cosford Nicholas
Published: December 2023

Autophagy is the process by which a cell maintains homeostasis by removing and recycling cytoplasmic debris. Autophagy provides lung cancer cells with essential nutrients for their increased proliferation and metabolism and aids in survival during anti-cancer treatments. CRISPR knockout or siRNA knockdown of the autophagy protein ATG13 inhibits autophagy and reduces viability of non-small cell lung cancer (NSCLC) cell lines. These data show that ATG13 is essential for autophagy and essential for lung cancer cell survival, making it a viable therapeutic target for cancer treatment. To identify compounds that degrade ATG13 and inhibit autophagy, we established a high-throughput screening (HTS) compatible Nano-Glo HiBiT assay where endogenous ATG13 was labeled on the C-terminus with HiBiT peptide via CRISPR. Optimization of this assay allowed for a signal to background Z’ factor of 0.71 and confirmed a positive control compound that induced up to 50% ATG13 degradation, thus indicating that this assay was suitable for compound screening. Currently, over 50,000 compounds from a variety of libraries have been screened in 1536-well format and 15 positive hits for ATG13 degradation have been identified. A series of counter-screens and secondary assays including In Cell Western Blot and Protein Thermal Shift assays will be used to confirm these hits. At the end of this testing funnel, we will have characterized novel compounds that degrade ATG13 and inhibit autophagy. These hits will provide a starting point for further chemical optimization to provide lead ATG13 degraders to evaluate in advanced preclinical proof-of concept studies of lung cancer.

Citation Format: Patrick M Hagan, Atoosa Emami, Hilarie Austin, Ian Pass, Allison Limpert, Douglas Sheffler, Nicholas Cosford. Discovery and development of novel ATG13 degrading compounds that inhibit autophagy and treat Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C040.
Abstract C043: Discovery of new drug combinations for metastatic colorectal cancer
Authors: In Eun-Jung , Tan Tao , Mouradov Dmitri , Sakthianandeswaren Anuratha , Au Amanda , Lowes Kym , Sieber Oliver
Published: December 2023

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Despite a range of therapeutic options, drug resistance remains a significant challenge in CRC treatment. Drug combination therapy offers an opportunity to enhance therapeutic responses. There are several combination drug options that have been used for CRC patients such as FOLFOX (5-FU + Oxaliplatin) and FOLFIRI (5-FU + Irinotecan). This study aimed to discover novel drug combinations for CRC by conducting high-throughput screening of 800 pharmacologically active compounds in combination with the three main chemotherapy backbones used in CRC treatment: 5-FU, SN-38, and Oxaliplatin. Initially, we performed a primary drug screen using 11 human CRC cell lines, resulting in the identification of 72 candidate drug combinations (24 candidates x 3 backbones) exhibiting synergistic effects. Subsequently, we validated these candidates in a subset of 5 cell lines. Among them, 24 drug combinations (8 candidates x 3 backbones) were selected and further evaluated in an expanded panel of 44 CRC cell lines to determine the spectrum of drug responses and identify potential mutation, gene and protein expression biomarkers, that could predict the efficacy of the drug combinations. We investigated the molecular mechanisms underlying the synergistic effects of final 4 candidates combined with chemotherapeutic agents. Chk1 inhibitor MK-8776 showed significantly higher synergy scores in combination with SN-38 in TP53 mutated cell lines, which was further validated by in vitro apoptosis analysis and in vivo xenograft models using TP53 wildtype and knockout isogenic CRC cell lines. MK-8776 abrogated SN-38-mediated G2 cell cycle arrest, resulting in an increased cytotoxicity. These findings highlight the potential of the combination treatment involving SN-38 and MK-8776 as a promising strategy for CRC patients with TP53 mutations.

Citation Format: Eun-Jung In, Tao Tan, Dmitri Mouradov, Anuratha Sakthianandeswaren, Amanda Au, Kym Lowes, Oliver Sieber. Discovery of new drug combinations for metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C043.
Abstract C044: Discovery and characterization of novel fragment binders of the VHR phosphatase as a drug target for cervix cancer
Authors: WU Jiaqian , Baranowski Marek R , Cosford Nicholas D.P. , Tautz Lutz
Published: December 2023

The dual-specificity phosphatase 3 (DUSP3), also known as Vaccinia H1-related phosphatase (VHR) was reported to exhibit elevated protein level in cervix cancer cell lines compared to normal keratinocytes implicating VHR as a novel target for cervical cancer therapeutic. VHR belongs to the superfamily of protein tyrosine phosphatases (PTPs), a large class of human enzymes that is notoriously difficult to target with small molecules. Recent studies have demonstrated some PTP functions as oncogenes to promote tumor development including SHP2 (PTPN11), PTP1B (PTPN1), and VHR (DUSP3). Fragment-based drug discovery (FBDD) has merged as an effective strategy to provide potent ligands, particularly for challenging drug targets such as PTPs. In this study, we present an NMR-based discovery platform designed to discovery fragment hits that exhibit binding affinity to VHR. Our platform employed fluorine NMR, a robust ligand-observed NMR method, to screen fragment library. Subsequent validation of confirmed hits was accomplished through a series of biophysical and biochemical experiments. Structural study such as X-ray crystallography provide binding mode of action of potent molecules with the VHR protein. Using this platform, we have successfully identified several potent fragments that displayed relative selectivity for VHR. Furthermore, We discovered novel ligand binding sites distinguished from the active site. Initial structure-activity relationship (SAR) studies were also conducted, enabling us to explore a set of validated fragment hits that serve as starting point for medicinal chemistry optimization. Moreover, our fragment discovery platform is a promising strategy for discovering potent and selective fragments targeting other PTPs, thereby offering potential therapeutic options for a boarder spectrum of cancer types.

Citation Format: Jiaqian WU, Marek R Baranowski, Nicholas D.P. Cosford, Lutz Tautz. Discovery and characterization of novel fragment binders of the VHR phosphatase as a drug target for cervix cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C044.
Abstract C054: PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition
Authors: Deng Ou , Li Xueli , Palve Vinayak C , Fang Bin , Reed Damon R , Rix Uwe
Published: December 2023

BACKGROUND: Ewing’s sarcoma (EWS) is a highly aggressive bone and soft tissue tumor with poor prognosis and an urgent need for novel therapies. We have identified that silencing of mono-ADP-ribosyltransferase PARP16 enhanced sensitivity of EWS cell lines to PARP1 inhibition. In this study, we explored the molecular mechanism of EWS vulnerability towards PARP16 targeting, alone and in conjunction with PARP1 inhibition, which may lead to development of PARP16-selective or dual targeting PARP1/16 inhibitors as new drugs for EWS.

METHODS: The effect of PARP16 on cell viability was analyzed using RNA interference with various siRNAs and doxycycline-inducible PARP16 shRNAs, as well as CRISPR/Cas9-mediated genomic deletion. The changes on total protein expression upon silencing of PARP16 with or without PARP1 inhibition was determined by mass spectrometry (MS). Protein interaction partners and downstream effectors of PARP16 were determined by Co-IP/MS and ADP-ribosylation proteomics, respectively.

RESULTS: Transient knockdown of PARP16 significantly reduced cell survival in EWS cell lines, particularly in combination with olaparib. Quantitative protein expression analysis by MS suggested doxycycline-induced shRNA-mediated PARP16 knockdown significantly reduced MYC protein expression in addition to PARP16 itself. Depletion of PARP16 in Cas9-expressing SK-N-MC EWS cells using different lentiviral sgRNAs further confirmed that targeting of PARP16 reduces cell survival and MYC expression, particularly in combination with PARP1 inhibition. Co-IP/MS using GFP-Trap agarose beads in mGFP/mGFP-PARP16 expressing cells suggested interaction of PARP16 with proteins that regulate post-translational modification of MYC in EWS cells. Further validation is ongoing.

CONCLUSION: Our data suggest that targeting PARP16 confers vulnerability to EWS cells either alone or in conjunction with PARP1 inhibition and that it is involved in regulation of MYC. Identification of novel PARP16 substrates and interaction partners is expected to produce molecular markers that enable the development of novel PARP16 inhibitors.

Citation Format: Ou Deng, Xueli Li, Vinayak C Palve, Bin Fang, Damon R Reed, Uwe Rix. PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C054.
Abstract C092: PRMT1 promotes pancreatic cancer development and resistance to chemotherapy
Authors: Ku Bomin , Eisenbarth David , Lim Dae-Sik
Published: December 2023

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer due to the lack of early diagnostic markers and effective therapeutic options. While epigenetic gene regulation is critically involved in the development of various types of cancer, its role in development of PDAC and the potential of therapeutically targeting epigenetic regulators remains underexplored. To identify a targetable epigenetic regulator, we utilized public datasets of pancreatic cancer and show that protein arginine methyl transferase 1 (PRMT1) is highly expressed in pancreatic cancer cells and that high expression of PRMT1 is a poor prognosis factor. Using loss of function studies in vitro and in vivo, we show that PRMT1 is essential for promoting cancer cell proliferation and tumorigenesis. We also demonstrate that the loss of PRMT1 relates to global changes in chromatin accessibility and gene expression pattern. Notably, we revealed that high PRMT1 negatively correlates with gemcitabine sensitivity and exhibit that pharmacological inhibition of PRMT1 in combination with gemcitabine had a greater anticancer effect on pancreatic cancer cells. Collectively, we suggest PRMT1 as a key factor of pancreatic cancer development and a potential candidate of chemotherapy.

Citation Format: Bomin Ku, David Eisenbarth, Dae-Sik Lim. PRMT1 promotes pancreatic cancer development and resistance to chemotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C092.