Induction Of Apoptosis Research Articles

Abstract 4671: Efficacy and mechanism of mebendazole for ovarian cancer therapy

Abstract Introduction: Ovarian cancer (OvCa) is the most lethal gynecological malignancy. Although most OvCa patients initially respond to frontline platinum-based therapies with approximately 80% of patients experiencing remission, approximately 75% of those women experience a recurrence with only approximately 50% of patients alive 5 years following diagnosis. There is a critical need to develop novel therapeutic agents or methods to overcome chemoresistance and toxicities experienced with conventional chemotherapy regimens used for OvCa patients. Repurposing drugs is a promising strategy to safely and more rapidly evaluate potentially effective treatment regimens. Over the past two decades, Mebendazole (MBZ), an anti-parasitic drug, has gained much attention in oncology due to its favorable biosafety profile and potent anti-cancerous activity seen in several human malignancies. Therefore, we examined the efficiency and mechanism of MBZ against OvCa. Methods: The in vitro efficacy of MBZ was evaluated in cisplatin-resistant (CPR) and parent (WT/sensitive) OVCAR-8 cell lines as well as in ascites samples collected from chemo-resistant OvCa patients by using 2D and/or 3D cell viability assay and invasion assay. The in vivo efficacy of MBZ was assessed in orthotopic animal models, utilizing CPR-OVCAR8 spheroids, and patient-derived xenograft (PDX) animal models with specimens from chemo-resistant ovarian cancer patients. Molecular mechanisms were investigated through western blot and immunofluorescence analysis. Result: Our results confirmed that MBZ efficiently decreased cell viability, spheroid size, and the invasion ability of OvCa cell lines and ascites samples. OvCa cells treated with MBZ demonstrated downregulation of proliferation markers and upregulation of apoptosis markers indicating inhibition of cell proliferation and induction of apoptosis. Mechanistically, MBZ inhibited the Wnt/β-catenin signaling pathway by downregulating the expression and nuclear localization of β-catenin and epithelial-mesenchymal transition (EMT) markers. Preclinical studies showed that MBZ at 50 mg/kg/day dose for 28 days significantly reduced tumor growth in PDXs and orthotopic tumor models without any evidence of toxicity. Conclusion: Collectively, our study strongly supports the therapeutic potential of MBZ against OvCa particularly, which warrants further clinical studies. Citation Format: Rajani Rai, Dhanamjai Penta, Debasish Dey, Doris M Benbrook, Lin Wang, Magdalena Bieniasz, Lauren E Dockery. Efficacy and mechanism of mebendazole for ovarian cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4671.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4752.

Abstract 551: Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has very limited treatment options. Xylopia Aethiopica (XA) is a common spice that is widely used in the regions of central and western Africa. It is known to have various pharmacological properties, one of which is anti-inflammatory activity. The anti-inflammatory effect may portend antiproliferative activity. We hypothesized that the extract of XA will exert antiproliferative activity against TNBC cells. The study aimed to establish the molecular mechanism underlying the antiproliferative activity of XA extract (XAE) against TNBC. The dried fruits of XA were crushed in a mixer and macerated using diethyl ether for 72 h. The macerate was subjected to vacuum and rotary evaporation to remove the organic solvent, and a stock solution was prepared in dimethyl sulfoxide (DMSO). Assessment of the efficacy of XAE was performed using serial dilutions (100 µg/ml, 50 µg/ml, 10 µg/ml, 5 µg/ml, and 1µg/ml) of the XAE stock solution, followed by in vitro testing against two TNBC cell lines (MDA-MB-231, and MDA-MB-468 cells). The cytotoxic effect of the XAE was then measured using the resazurin dye assay. XAE resulted in a concentration-dependent cell death in both cell lines. At the highest concentration (100 µg/ml), XAE induced cytotoxic cell death of 89% and 91% in MDAMB231 and MDAMB468 cells, respectively. This was consistent with IC50 values of 32.95 ± 1.32 µg/ml (MDAMB231) and 20.83 ± 1.05 µg/ml (MDAMB468). At the molecular level, western blot analysis of mitogenic markers associated with MAPK oncogenic signaling pathway demonstrated the inhibition of EGFR and AKT protein levels after a 48-h treatment. Acridine orange-ethidium bromide (AO/EB) staining showed a concentration-dependent induction of apoptosis in both cell lines. Flow cytometric analysis was performed to quantify the apoptotic cells. A significant increase of apoptotic cells was observed in treatment groups as compared to control after 72 h. Apoptotic induction was associated with increased p53 protein levels after 48 h treatment compared to control. The study results underscore the anticancer effects of XAE in TNBC cells mediated through induction of apoptosis. Citation Format: Shailvi Soni, Shail Rakesh Modi, Terrick Andey. Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 551.

Abstract 650: Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer

Abstract Triple-negative Breast cancer (TNBC) is characterized by the absence of ER, PR, and HER-2 receptors and is associated with poor prognosis, early relapse, and distant metastasis. In neoadjuvant setting, most patients (~50-70%) with TNBC do not achieve complete remission. Currently, there is no effective targeted therapy for TNBC patients. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. PIM3 is a serine/threonine kinase and is upregulated in leukemia and various solid cancers. Although PIM1 has been shown to play a critical role in cell proliferation in cMyc-enriched TNBC tumors, the role of PIM3 in TNBC is not known. To determine the clinical significance of PIM3 in TNBC patient prognosis and progression, we performed a Kaplan-Meier analysis in the TCGA TNBC patient database. We found that PIM3 expression is significantly associated with shorter patient survival in TNBC patients (n=77, p=0.0088). PIM3 protein is highly expressed in TNBC cells compared to normal breast epithelium and other ER+ and HER2+ breast cancer subtypes. Inhibition of PIM3 by siRNA significantly suppressed TNBC cell proliferation, migration, and invasion of TNBC cells including MDA-MB-231, MDA-MB-436, and BT20 cells. Lenti-based PIM3 overexpression induced TNBC cell proliferation, invasion, and migration. Non-biased reverse phase protein array (RPPA) assay and Western blot analysis showed that PIM3 inhibition markedly reduced the expression of EF2 Kinase protein, and we found it to be associated with shorter patient survival and poor prognosis. Immunoprecipitation of PIM3 indicated that it forms a heterodimer with EF2K whose inhibition by siRNA also suppressed TNBC cell proliferation, migration, invasion, and tumor growth. Furthermore, PIM3 inhibition by siRNA significantly enhanced the antiproliferative effect of Doxorubicin, which is one of the first-line chemotherapeutics used in patients. Moreover, in vivo therapeutic delivery of PIM3 siRNA loaded albumin nanoparticles (ALNPs-PIM3 siRNA) markedly delayed tumor growth of two different TNBC tumor xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Analysis of tumors by IHC after 4 weeks of treatment demonstrated that ALNP-PIM3 siRNA treatment led to inhibition of intra-tumoral proliferation and induction of apoptosis. Currently, we are conducting an in vivo study in TNBC tumor models by combining ALNP-PIM3 siRNA therapy with doxorubicin. Overall, our studies suggest that PIM3 is a marker for poor patient survival that drives TNBC tumor growth and progression and a novel potential molecular target in TNBC. Citation Format: Pinar Atalay, Goknur Kara, Rumeysa Ozyurt, Nermin Kahraman, Bulent Ozpolat. Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 650.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3229.

Abstract 568: Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models

Abstract Introduction HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology that includes four isoforms (JMa-CYT1/2, and JMb-CYT1/2) and tissue specific oncogenic or tumour suppressor roles. In estrogen receptor positive (ER+) breast cancer (BC), HER4 isoforms can either act as a co-regulator of ESR1-mediated gene expression by localising to the nucleus, or activate apoptosis by localising to mitochondria. There is very little known about HER4 function in cutaneous melanoma, despite cutaneous melanoma showing the highest HER4 genomic alteration frequency (17.34%) across different cancers (cBioPortal). Due to the interplay between estrogen signalling and HER4 isoforms in ER+BC, this study investigates the RNA expression of HER4 isoforms along with hormone and nuclear receptors associated with 17β-estradiol stimulation in ER+BC and cutaneous melanoma in vitro models. Material and Methods CAMA1 (ER+BC) and SKMEL24 (cutaneous melanoma) cell lines were serum starved for 4h prior to stimulation with 17β-estradiol (0.5nM, 1nM, 5nM and 10nM). RNA was extracted from cells after 24h using TRIzol, followed by qRT-PCR to assess the expression of HER4 isoforms, the hormone receptors estrogen receptor 1 (ESR1/ER-alpha), estrogen receptor 2 (ESR2/ER-beta), progesterone receptor (PGR/PR), G protein-coupled estrogen receptor 1 (GPER1) and the nuclear receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). Cell proliferation and caspase3/7 apoptosis analysis were performed at 24/48/72h in the 17β-estradiol stimulated cells by using the IncuCyte Live Cell Analysis Instrument (Sartorius). SN-38 (150nM) was used as positive control for apoptosis induction. Two-way ANOVA was used for the statistical analysis, p<0.05 was significant. Results The qRT-PCR analysis showed that upon stimulation of CAMA1 with 1nM of 17β-estradiol there is a numerical shift in the CYT-1/CYT-2 ratio towards CYT-2 (not statistically significant, p>0.05) and a significant increase in PGR (p= 0.0027) and RAR-A (p= 0.0248) expression compared to control. CAMA1 stimulated with any concentration of 17β-estradiol showed an increase in proliferation compared to the control. The stimulation of SKMEL24 with 5nM of 17β-estradiol showed an increase in JM-a (p=0.0075) and CYT-1 (p=0.0271) HER4 isoforms, GPER1 (p= 0.0286), and a dose-dependent increase in RARA (p=0.0011) compared to control, even in the absence of ESR1, ESR2 and PGR. SKMEL24 stimulated with 17β-estradiol showed a decrease in proliferation compared to control, but no apoptosis induction was found. Conclusion These findings suggest potential interplay between 17β-estradiol and HER4 isoforms leading to the activation of intracellular pathways that could influence tumour progression/suppression in melanoma. Further studies are needed to clarify the specific receptor interactions and pathways involved. Citation Format: Marta Valenti, Georg F. Bischof, John P. Crown, Denis M. Collins. Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 568.

Abstract 4579: DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells

Abstract Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) are overexpressed in cancer cells associated with dysregulated gene expression and tumor progression. DNMT1 partnered with Class I HDAC as complex to inhibit gene transcription. Several pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. More selective HDAC inhibitors need to be developed. Psammaplin A (PsA), a symmetrical bromotyrosine-derived disulfide dimer isolated from the Psammaplysilla sponge, is a selective Class I HDAC inhibitor. We tested its cell growth inhibition and apoptosis induction in 12 hematological malignant cell lines with 8 lines sensitive (more than 50% growth inhibition at 1 μM) and 4 lines insensitive (less than 20% growth inhibition at 1 μM). PsA induces acetylation of H3, but not α-tubulin, supporting the Class I HDAC inhibition selection. In the sensitive cell lines, PsA induces apoptosis with down-regulation of c-Flip and upregulation of NOXA as well as DNA damage detected by γ-H2A.X. RNA sequence analysis results confirm the regulation of PsA on NOXA, c-Flip and p21 gene expression. The four insensitive cell lines express high levels of DNMT1 with resistance to apoptosis induction. Silencing DNMT1 with siRNA sensitizes PsA-induced apoptosis with induction of NOXA and γ-H2A.X. Azacytidine, the DNMT1 degrader, in combination with PsA induce synergistic cell growth inhibition and apoptosis in vitro. PsA (20 mg/kg) plus Azacytidine (2.5 mg/kg) synergistically inhibit tumor growth in insensitive mantle cell lymphoma Jeko-1 xenografts, without detected toxic signs in organ pathological analysis. We find a new mechanism of DNMT1 controlling the sensitivity of Class I HDAC inhibitor PsA and provide a rationale of PsA with azacytidine for combination therapy. Citation Format: Yu Bao, Dan Liu, Zi Wu, Yumei He, Ping Gong, Samuel Waxman, Yongkui Jing. DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4579.

Abstract 3195: SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in xenograft models

Abstract Background: Current clinical HER2-targeting ADCs (i.e. DS-8201) have shown promising clinical efficacy in HER2 expressing or mutant cancer patients. However, most of them will ultimately experience disease progression due to different mechanisms. Herein, we presented a novel HER2 ADC, SHR-4602, which demonstrated good in vitro and in vivo anti-tumor activities and could overcome the resistance caused by HER2 ADC including DS-8201 analogue (synthesized using DS-8201 structure) and SHR-A1811 (composed of trastuzumab, a cleavable linker, and a Topo I inhibitor payload, in phase III clinical trials) in xenograft models. Method: We used a stable enzyme-cleavable linker technology to conjugate the HER2 ADC using the amino acid sequence of pertuzumab and ER300 (eribulin derivative). The in vitro biological and anti-tumor activities were assessed by a series of assays. The in vivo anti-tumor efficacies were evaluated in the JIMT-1 (HER2 low expressing) as well as the SHR-A1811 and DS-8201 analogue induced JIMT-1 resistance (caused by treating with SHR-A1811 followed by DS-8201 analogue for a long-term) xenograft models in BALB/c nude mice. Results: Through the SPR assay, SHR-4602 exerted single nanomolar binding affinity to human and rhesus monkey HER2, but no binding activity against rat HER2. It demonstrated varying inhibitory activity on the proliferation of cancer cell lines with various HER2 expression levels, with the IC50 at the range of 0.007 nM to 8.21 nM. SHR-4602 showed highly efficient internalization in NCI-N87 HER2 expressing cells and could be translocated to the lysosome followed by releasing ER300. The cytotoxic activity of SHR-4602 was predominantly mediated by released ER300 through microtubule dynamics inhibition, G2/M arrest and apoptosis induction. Furthermore, SHR-4602 showed a strong in vitro bystander effect in the co-culture of HER2 positive/negative cells. SHR-4602 showed significant anti-tumor efficacy in JIMT-1, SHR-A1811 and DS-8201 analogue both-resistant JIMT-1 xenograft models. In SHR-A1811 and DS-8201 analogue both-resistant xenograft models, treatment with 10 mg/kg SHR-A1811 or DS-8201 analogue exerted negligible anti-tumor activity (TGI% < 17%), whereas SHR-4602 at 2.5, 5 and 10 mg/kg significantly inhibited the tumor growth (p < 0.001, vs. vehicle), with TGI% of 84.96%, 101.64% and 113.11% on Day 42, respectively, which indicated a superior anti-tumor efficacy against SHR-A1811 and DS-8201 analogue both-resistant xenograft models. Conclusion: SHR-4602 represents a new generation HER2-targeting ADC to deliver a potent cytotoxin to HER2 expressing tumor cells and may be more efficacious to the tumors in which current anti-HER2 therapies are ineffective. SHR-4602 is now in the phase I clinical development stage for advanced malignant solid tumors (NCT05819684). Citation Format: Yunan Tian, Xing Sun, Changyong Yang, Cheng Liao. SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3195.

Abstract 40: Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit

Abstract Adoptive cell transfer of chimeric antigen receptor (CAR) engineered T and NK cells has revolutionized the treatment of many B-lymphoid malignancies; however, limited persistence of infused lymphocytes can restrain the full potential of this approach. Apoptosis induction through the FAS ligand/FAS pathway is a key negative regulator of lymphocyte survival. To define which cellular subsets express FASLG, the gene encoding FAS ligand, we generated a single-cell transcriptomic atlas from 37 patients with diverse cancer types. We discovered that FASLG expression is highly constrained and limited primarily to T and NK cells while stromal and tumor cells express minimal to no FASLG. Multiplex RNA in situ hybridization of clinical samples from patients treated with anti-CD19 CAR-T cells confirmed these results and revealed significantly higher FASLG content in CAR-expressing compared with non-CAR expressing T cells. Based on these findings, we hypothesized that FAS ligand expression by CAR+ T and NK cells will engage FAS+CAR+ cells to induce apoptosis and limit cellular persistence. To test this hypothesis, we performed competitive fitness assays using two phenotypically discernable populations of human CAR-T and CAK-NK cells that are either responsive or unresponsive to FAS-signaling through expression of a FAS dominant negative receptor (ΔFAS). ΔFAS+ and ΔFAS− CAR-expressing cells were co-infused 1:1 into tumor bearing hosts and persistence of each population was quantified. For both CAR-T and CAR-NK, we measured significant enrichment in ΔFAS-expressing cells across multiple tissues, demonstrating that FAS-signaling regulates CAR+ cell survival in a cell-intrinsic manner. Knock out (KO) of FASLG using CRISPR/Cas9 prevented population skewing while KO of the AAVS1 control locus had no effect, indicating that CAR-derived FASLG is sufficient to restrain the persistence of transferred cells. By contrast, we discovered that FASLG was dispensable for CAR-T and CAR-NK mediated tumor killing both in vitro and in vivo. Finally, using two different tumor models, we found that adoptive transfer of CAR-T or CAR-NK cells that co-express ΔFAS resulted in superior antitumor efficacy compared with control cells that express the CAR alone. Taken together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS/FAS ligand auto-regulatory circuit. Further, they indicate that the therapeutic benefit of a FAS-dominant negative receptor is broadly applicable and agnostic of whether FASLG is expressed by cells in the tumor microenvironment. Citation Format: Fei Yi, Tal Cohen, Paul Zumbo, Razan Eltilib, Hannah Arkin, Michael Gormally, Christopher S. Hackett, Korbinian Kropp, Smita Chandran, Jae H. Park, Doron Betel, Christopher A. Klebanoff. Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 40.

Abstract 3207: Chemical fabrication and characterization of a novel nanoparticle of the histone deacetylase (HDACi) romidepsin

Abstract Introduction: Histone Deacetylase inhibitors (HDACi) are important drugs for the treatment of patients with PTCL (peripheral T-cell lymphoma). Romidepsin (R), a potent Class I HDACi, is given intravenously, weekly for three to four consecutive weeks. R has a half-life of three hours and is extensively protein bound. The indication for R in PTCL was recently withdrawn due to a failed Phase 4 study. Given the need for new drugs in PTCL, and the importance of epigenetically targeted drugs for the disease, we developed a first-in-class nanoparticle of R (NR) deploying a proprietary nanochemistry platform. Methods: We modified the bulk nanoprecipitation method with FDA approved and Generally Recognized as Safe (GRAS) materials, specifically including PEGylated amphiphilic diblock copolymers, to develop NR combinatorially. By combining a drug and polymer solution with an anti-solvent, we produced core-shell structured NR particles with “stealth” properties. To achieve high R loading, stability, purity, scalability, and reproducibility, we devised a multi-pronged optimization system by modulating parameter sets in tandem. We determined the optimal parameters for the "lockdown" formulation. We purified NR by using centrifugal filters and determined stability by storing at 4°C. We used Cryo-electron microscopy (cryo-EM), Dynamic Light Scattering (DLS), and Liquid Chromatography-Mass Spectrometry (LC-MS) to determine the morphology, size, PDI, and drug concentration of NR. We developed fluorescent-NR with DiO dye to test bioavailability. We tested NR in T-cell lymphoma cell lines for histone acetylation and apoptosis induction. Furthermore, we investigated the in vivo pharmacokinetics and toxicity of NR in PTCL xenograft mouse model and quantified the drug by LC-MS. Results: The physiochemical analysis of NR demonstrated that the particles were spherical, with a hydrodynamic diameter of 46 ±5 nm and PDI of 0.15 - 0.2. NR had a negative zeta potential and an exceptional drug encapsulation efficiency of over 50% (>500 µg/mL). NR exhibited high stability with respect to size, PDI and drug concentration for over two years. Flow cytometry and western blot analyses indicate NR induces apoptosis and histone acetylation in a time and dose-dependent manner, equivalent to or better than that observed for R. NR’s AUC increased 25-fold when administered intravenously. NR had superior tolerability, and DiO-NR confirmed superior biodistribution in tumor in a PTCL xenograft mouse model. Conclusion: We developed a cutting-edge approach with minimum excipients, which enabled NR scale up with batch-to-batch reproducibility and high quality co-packaging of fluorescent dye and drug. NR improved R’s pharmacokinetic profile and efficacy in pre-clinical xenograft mice models. Our versatile technology enables Chemistry Materials and Controls products, an essential step in clinical product development. Citation Format: Samir Zuberi, Deepthi P. Damera, Ipsita Pal, Susan Walker, Ramesh Katla, Colin Haws, Akhil Gajjala, Andrea Joyner, Kallesh D. Jayappa, David J. Feith, Todd E. Fox, Thomas P. Loughran, Owen A. O'Connor, Anuradha Illendula. Chemical fabrication and characterization of a novel nanoparticle of the histone deacetylase (HDACi) romidepsin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3207.

Abstract 7135: Preclinical effects of Tumor Treating Fields (TTFields) with PARP inhibitors in ovarian cancer

Abstract Purpose: Among gynecological malignancies, ovarian cancer is the leading cause of mortality. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent standard-of-care treatment for selected cases of advanced epithelial ovarian cancer. PARPi exhibit sensitivity in homologues recombination deficient (HRD) tumors, in which there are mutations in the Fanconi Anemia (FA)-BRCA pathway, while about 75% of ovarian cancer patients are homologues recombination proficient (HRP). TTFields are non-invasive electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Recent research showed that TTFields induce an HRD-like state (also known as BRCAness state) in various cancer types. The current study aims to investigate the impact of TTFields applied together with PARPi in ovarian cancer preclinical models. Methods: In vitro, A2780 (HRP), OVCAR3 (HRD), and A2780cis (platinum-resistant) ovarian cancer cells were treated with TTFields (1 V/cm RMS, 200 kHz, 72 h), alone or with addition of PARPi (olaparib or niraparib, at various concentrations). Efficacy was measured by analyzing cell count, colony formation, and apoptosis induction. The overall effect was calculated by multiplying cell count with colony formation. TTFields-treated cells were evaluated for the expression of FA-BRCA pathway proteins and for formation of γH2AX, a DNA damage marker, using Western blot and fluorescent microscopy, respectively. In vivo, ID8-fLuc (HRP) cells were inoculated intraperitoneally to 6-8-week-old C57BL/6 mice. Seven days post inoculation, mice were treated with sham-vehicle, TTFields, olaparib (50 mg/kg), or both over a period of four weeks. Tumor growth was analyzed using bioluminescent imaging at treatment cessation, and survival analysis was performed. Results: TTFields application resulted in reduced cell count, increased overall effect, and increased apoptosis in vitro. When TTFields were applied together with PARPi, a high and moderate synergistic interaction was shown respectively the BRCA wild-type A2780 cells and the platinum-resistant A2780cis cells. However, in the BRCA mutant OVCAR-3 cells the effect was additive. A downregulation of FA-BRCA protein expression was confirmed after TTFields treatment of the ovarian cells, with consequent DNA damage. In the tumor-bearing mice, TTFields and olaparib co-treatment resulted in reduced tumor volume and a survival benefit relative to the monotherapies and to control-treated mice. Conclusions: The data suggest potential advantages for TTFields concomitant with PARPi in ovarian cancer, even without underlying BRCA mutations - consistent with the BRCAness state induced by TTFields - and also suggests benefits in platinum-resistant ovarian cancer. Citation Format: Yani Berckmans, Hila Ene, Kerem Ben-Meir, Antonia Martinez-Conde, Roni Monin, Bieke Van den Ende, Sara Van Mechelen, Roni Frechtel-Gerzi, Hila Gabay, Rotem Engelman, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti, Ignace Vergote, An Coosemans. Preclinical effects of Tumor Treating Fields (TTFields) with PARP inhibitors in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7135.

Abstract 5720: Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models

Abstract Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma. Mycosis fungoides and Sézary syndrome (MF/SS) are the most common subtypes in CTCL. Early-stage MF/SS has a favorable prognosis. The advanced-stage CTCL has limited treatment options and a median survival of <5 years, highlighting the urgent need to develop targeted therapies in CTCL. Preclinical studies of CDK9 inhibitors have shown anti-tumor activity in various subtypes of hematologic malignancies. Several CDK9 inhibitors have entered clinical trials for lymphomas and leukemias. However, whether targeting CDK9 in CTCL is a therapeutic option is little known and has resulted in CTCL not being included in ongoing clinical investigations of CDK9 inhibitors. AZD4573 is a highly selective and potent CDK9 inhibitor currently under clinical investigation for Hodgkin and non-Hodgkin lymphomas. It was shown that AZD4573 induces tumor cell death through depletion of MCL-1. Overexpression of MCL-1 has been shown in CTCL cell lines and skin biopsy specimens of CTCL patients. MCL-1 expression appears to be particularly elevated in patients with advanced stage disease. Here, we investigated the ex vivo anti-tumor activity of AZD4573 using patient-derived CTCL xenograft models (PDXs). Primary tumor cells harvested from CTCL PDX mice were activated with CD2/CD3/CD28 beads and incubated in hIL-2 and hIL-7 at an optimal cell density. Subsequently, cells were exposed to AZD4573 at various concentrations for 8 hr, then treated cells were harvested, washed, and placed in fresh media for another 48 hr. Growth inhibition was measured by the Real-Time Glo cell viability assay, and apoptotic activity was assessed by flow cytometry analysis of Caspase 3/7 activity and Annexin V + propidium iodide (PI) double staining. Additionally, we conducted RNA-seq analysis for MCL-1 expression in CTCL PDX primary tumor cells. AZD4573 inhibited cell proliferation in a dose-dependent manner in primary tumor cells from three CTCL PDX mice (PRS-1, PRS-2 PRS-3), with GI50 of 43.5 nM, 175.4 nM, and 113.5 nM, respectively. Furthermore, AZD4573 significantly increased caspase 3/7 activity and apoptosis in PRS-1 and PRS-3, but not PRS-2 primary tumor cells. In addition, our RNA-seq analysis showed that compared with normal controls (normal PBMC from healthy donors), the MCL-1 gene expression is increased 10-fold (P-value <0.0001) in PRS1 cells. The MCL-1 gene expression in PRS2 and PRS3 cells is the same as normal control. Furthermore, PRS-1, which has the highest MCL-1 expression level, is most sensitive to AZD4573. In conclusion, AZD4573 demonstrated dose-dependent growth inhibition and apoptosis induction in primary CTCL tumor cells. In addition, the expression level of MCL-1 appeared to be correlated with the ex vivo anti-tumor activity of AZD4573. Our findings provide a rationale for clinical investigation of targeting CDK9 in CTCL. Citation Format: Yan-Jin Liu, Chi-Heng Wu, Laura Pincus, Weiyun Z. Ai. Targeting CDK9 in cutaneous T-cell lymphoma using patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5720.

Abstract 4617: Fisetin, a multi-kinase inhibitor enhances chemotherapeutic sensitivity in head and neck cancer

Abstract Background: The limitations of chemotherapeutic applications are often imposed by dose-related toxicity and the emergence of therapeutic resistance. Fisetin, a naturally occurring flavonoid, has demonstrated anticancer properties in various cancer types. Despite its proven effectiveness against cancer, the molecular target of fisetin remains unknown. Methods: Our study focused on understanding how fisetin and chemotherapy work together to inhibit the growth and survival of human HNC. We conducted in silico, in vitro and in vivo investigations. We employed target prediction, kinase profiling, transcriptome analysis and tumor xenograft to identify the chemosensitivity efficacy and mechanism of fisetin. Results: In-silico target prediction suggested that fisetin might target multiple kinases. Based on this lead, we have screened fisetin for different kinase inhibition activities and found that it inhibits multiple CDKs including CDK1, 2, 4, 5 and 6 at nanomolar concentration. To test its cellular activity, we have selected the head and neck cancer (HNC) model and found that fisetin treatment strongly inhibited cell proliferation and arrested the HNC cells in G1 phase followed by apoptotic cell death. In tumor xenograft experiment, we found that oral fisetin treatment inhibited the FaDu and SAS tumor growth in the athymic mouse model. Transcriptome analysis on FaDu and SAS cell lines indicated that fisetin treatment significantly modulates pathways associated with cell cycle progression and regulation. These pathways include cellular assembly, DNA replication, cell death, and cell morphology. Pretreatment of HNC cells with fisetin improved the effectiveness of chemotherapy drugs including cisplatin, 5-fluorouracil, and docetaxel. Consistent with this, fisetin pre-treated cells showed elevated levels of cellular and mitochondrial ROS followed by the activation of caspases and apoptosis induction when compared with that in control cells after cisplatin, 5-FU, or docetaxel treatment. Conclusion: These results imply that fisetin has the potential to be utilized as a multi-kinase inhibitor in cancer cells. The study revealed that fisetin has a strong potential for increasing the sensitivity of cancer cells to chemotherapy. Citation Format: Dhanir Tailor, Deepali Mishra, Sanjay V. Malhotra, Rana P. Singh. Fisetin, a multi-kinase inhibitor enhances chemotherapeutic sensitivity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4617.

Abstract 7589: The novel complex delivery system of C-phycocyanin-loaded klebsiella pneumoniabacterial ghost induces apoptosis and sensitizes non-small cell lung cancer cell lines to doxorubicin

Abstract Lung cancer is a significant cause of cancer-related deaths worldwide, with a high incidence rate, poor prognosis, aggressiveness, and a lack of effective treatment options. The present study has brought together two FDA-approved drugs with antineoplastic properties to create a complex that promises to revolutionize the treatment of Non-Small Cell Lung Cancer (NSCLC). By combining C-Phycocyanin extract (PCE), a pigment-protein complex synthesized by blue-green microalgae platensis, with the immunostimulatory doxorubicin (DOX) loaded Bacterial Ghosts (BGs), the present study aims to achieve enhanced dual-chemotherapy and remarkable inhibition of DOX-chemoresistance. The BG delivery system uses purified bacterial cell evacuation and safe confirmed Klebsiella pneumoniae BGs (KPBG) for safe and effective delivery, overcoming issues with resistance and poor delivery. The complex delivery system of KPBG-PCE/DOX was tested on two NSCLC cell lines, wild-P53, A549, and mutant-P53, NCI/H358, respectively. Our data showed that the loaded combined treatment significantly induced morphological changes with signs of apoptosis induction in the tested cell lines compared with the control. The WST-1 assay was used to evaluate cell proliferation. Data indicates that PCE and DOX have a synergistic effect at low concentrations when loaded on KPBGs for a constant count of BGs CFU/ml after 48 h of treatment. The viability inhibition and apoptosis induction of A549 and NCI/H358 cells were amplified in the combined treatment of 48 h compared to the control or either each drug alone. At the protein levels, our data showed activation of caspase-3, upregulation of BAX, and downregulation of Bcl-2, Bcl-XL, m-TOR, S6k, oncogenic β-catenin, and cyclin-D1 protein expression levels after treatment with (KPBG-PCE/DOX) for 48 h, indicating apoptotic induction and modulating oncogenic signaling pathways. In conclusion, the current study presents a promising outcome of a synergistic delivery system of KPBG-PCE/DOX that can inhibit lung cancer cell line proliferation, induce apoptosis, and increase cell sensitivity to DOX. The KPBG delivery system offers an appealing approach for a successful combination of targeted therapy for lung cancer. Citation Format: Basma Adel Elsayed Abdelaziz, Hoda Eid Mahmoud, Omayma Mohamed Sadek, Ahmed Samir Sultan. The novel complex delivery system of C-phycocyanin-loaded klebsiella pneumoniabacterial ghost induces apoptosis and sensitizes non-small cell lung cancer cell lines to doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7589.

Abstract 518: The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation

Abstract Richter transformation (RT), characterized by the transformation of chronic lymphocytic leukemia (CLL) to an aggressive large-cell lymphoma, remains a challenge for the therapy. Genetic aberrations, such as Myc activation, TP53 abnormality, NOTCH1 gene mutation or loss of CDKN2A are associated with uncontrolled RT cell proliferation and escaping of apoptosis. Among those changes, gain of Myc activity, found in around 70% of RT, plays a nexus role in the pathogenesis of RT. As a transcription factor, Myc activation has a profound effect on cell survival, proliferation, adhesion and metabolism. Because of its essential role in lymphoma transformation and aggressiveness, Myc is a vital target for therapy toward RT. Fadraciclib (CYC065) is a second-generation CDK inhibitor with selective potency toward CDK2 and CDK9. We have shown previously that by inhibiting CDK9-mediated transcription, fadraciclib reduced expression of the short-lived anti-apoptotic protein Mcl-1 to initiate apoptosis in primary CLL cells. Like Mcl-1, Myc is a quintessential example of an oncogene with rapid turnover in both its mRNA and protein, therefore a promising target of CDK9 inhibition. Thus, we proposed that fadraciclib would target both the survival and proliferation pathways of RT pathogenesis through collaborative inhibition of Mcl-1, Myc and CDK2. First, our data showed that fadraciclib effectively induced apoptosis in the RT cell line HPRT1, as well as the lymphoma cell lines with Myc amplification, including Raji, Ramos and SU-DHL-2. The IC50 for apoptosis was 0.3 µM at 24h in the HPRT1 cells, which is less than the average IC50 for the primary CLL cells (0.8 µM). The IC50 for the lymphoma lines ranged from 0.8 µM to 1.3 µM. Immunoblots showed reduction of RNA Pol2 phosphorylation by fadraciclib, consistent with the inhibition of CDK9. As a result of loss of RNA Pol2 activity and pausing of transcription, there was a clear reduction of both Mcl-1 and Myc mRNA, measured by real-time RT-PCR, that was associated with the reduction of their protein levels. CDK2 was also inhibited by fadraciclib, shown by the reduction of the phosphorylation of both RB and NPM. Second, Fadraciclib inhibited cell proliferation of the HPRT1 cells at an IC50 of 0.25 µM measured by the CellTiter-Glo assay. The IC50 for growth inhibition in the lymphoma lines ranged from 0.5 to 0.7 µM. CFSE tracing demonstrated halting of cell division upon fadraciclib incubation. Cell cycle analysis by Click-iT EdU showed reduction of S-phase population and arresting of cells in both G1 and G2. We are currently working on dissecting the contributions of inhibiting Myc, Mcl-1 and CDK2 individually and together, in apoptosis induction and proliferation blockade. Taken together, these data suggest that fadraciclib is active in RT cells and Myc-dependent lymphoma cell lines and support expansion of its clinical testing in patients with RT. Citation Format: Rong Chen, Yuling Chen, Ping Xiong, William Plunkett. The CDK2/9 inhibitor fadraciclib is active in Richter transformation and lymphoma cell lines by targeting both cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 518.

Abstract 5987: Targeting the spliceosome in high-risk B-cell acute lymphoblastic leukemia

Abstract The outcome for children with high-risk B-cell acute lymphoblastic leukemia (B-ALL) is poor. Disease relapse is speculated to be due to leukemia cells escaping treatment. Our group discovered a unique cell subpopulation in B-ALL that has the capacity to initiate leukemia and is resistant to treatment. Transcriptome studies of this cell population highlight a unique RNA regulation process, specifically RNA splicing. In B-ALL, dysregulated splicing is reported to be associated with drug resistance and disease relapse. SF3B1 is a core component of the spliceosome and an essential protein in the RNA splicing process. SF3B1 inhibition is therapeutic in many cancers. In our current studies, we investigated the therapeutic potential of pladienolide B (Plad-B), an SF3B1 inhibitor, in B-ALL. SF3B1 protein expression was significantly higher in cell lines (Reh and JM1) and 28 primary B-ALL samples (14 each for standard-risk and high-risk), regardless of the risk group, than in normal B-cells (NBs) and hematopoietic stem cells (HSCs). Plad-B showed significant dose-dependent cytotoxicity in the cell lines with IC50 of 1.2nM and 0.6nM, respectively, and three harvested high-risk patient-derived xenograft (PDX) samples with almost the same IC50 as the cell lines. Plad-B did not show cytotoxicity in NBs and HSCs at the same tested concentrations. In vivo efficacy of Plad-B was tested using an Reh xenograft mouse model and a high-risk PDX model. Plad-B, as a single drug treatment, significantly prolonged survival in both models (p<0.01). G2/M cell cycle arrest and apoptosis induction were observed at 24 hours after Plad-B treatment in Reh and JM1. Splicing events were examined by RNA-seq in the treated cells at different time points (15 min, 30 min, and 60 min). Plad B demonstrated rapid splicing inhibition as early as 15 min post-treatment and, at 60 min, splicing was inhibited in 1,669 genes, including apoptosis-associated genes. 2,625 differential splicing events were observed in these genes with ~96% from intron retention and exon skipping. Furthermore, 202 genes showed significant changes in their expression over this time frame. Most of the genes were rapidly downregulated in the treatment group compared to the control, and some were slowly downregulated. There were also some genes which were upregulated, either rapidly or slowly, compared to the control. We demonstrated that Plad-B induced short pro-apoptotic spliced isoforms, instead of anti-apoptotic forms, in BCL2L1 and MCL-1, as early as 60 min after treatment. In conclusion, these data demonstrated the therapeutic potential of SF3B1 inhibition in high-risk B-ALL. Plad-B rapidly inhibited splicing in many genes and downregulated the anti-apoptotic forms of apoptosis-associated genes, leading to cell apoptosis. In future studies, we will identify the downstream targets of Plad-B and further investigate the mechanism of rapid apoptosis induction by SF3B1 inhibition. Citation Format: Yuki Murakami, Hiroaki Konishi, Clifford Tepper, John McPherson, Noriko Satake. Targeting the spliceosome in high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5987.

Abstract 4686: Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer

Abstract Breast cancer (BC), the most prevalent malignancy in women, ranks fourth in cancer mortality as of 2022. Mutations in the BRCA genes significantly elevate BC risk and are associated with markedly reduced survival compared to non-carriers. These genes are crucial in homologous recombination, a mechanism essential for error-free DNA repair. Despite advancements, treatment options for BRCA mutation-driven cancers remain limited. Synthetic lethality (SL) has emerged as a promising strategy, with poly(ADP-ribose) polymerase inhibitors (PARPi) being the first developed drugs based on this principle. However, PARPi's effectiveness is limited, as nearly 40% of BRCA-mutated BCs are unresponsive, and resistance often develops in initial responders. Therefore, novel mutation-specific targeted therapies are urgently needed. In this study, we employed the novel computational method 'Mining Synthetic Lethals (MiSL)' to analyze TCGA pan-cancer primary tumor data, identifying ART3 as a potential SL partner of the BRCA1 germline mutation. Our approach focused on the intersection of genetic interactions and cancer vulnerabilities, providing a unique insight into SL relationships. siRNA-mediated silencing of ART3 in BRCA1-mutated cells consistently led to decreased cell viability, apoptosis induction, and G2/M cell cycle arrest. Following stable transfection with an inducible ART3 shRNA construct, we confirmed significant reduction in cell viability in BRCA1-deficient cells through colony formation assays. RNA sequencing of ART3 knockdown cells revealed transcriptional changes in genes involved in cell cycle progression, apoptosis, and growth and proliferation pathways compared with control cells. Immunofluorescent staining revealed increased DNA damage foci with decreased DNA repair in ART3 knockdown BRCA1-deficient cells. In conclusion, our study identifies ART3 as a novel SL partner of the BRCA1 mutation in BC. This discovery paves the way for developing new therapeutic strategies for BRCA1-mutated breast cancers and novel chemoprevention approaches for individuals carrying germline BRCA1 mutations. Future research will focus on further elucidating the molecular mechanisms underlying this SL interaction and exploring its therapeutic potential in clinical settings. Citation Format: Hannah Neiger, Xinyu Pei, Anh Nguyen, Wenjia Wang, Emily L. Siegler, James Zhou, Kristie Lau, Alexandra Tan, Yihui Shi. Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4686.

Abstract 6579: DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models

Abstract Background: Tumor-associated mucin-1 (TA-MUC1) is a tumor-specific transmembrane glycoprotein with aberrant glycosylation. It is highly expressed in various human epithelial cancers, making it an attractive target for cancer therapy. DS-3939a is a TA-MUC1 targeting antibody-drug conjugate (ADC) structurally composed of a humanized anti-TA-MUC1 antibody, an enzymatically cleavable tetrapeptide-based linker, and a DNA topoisomerase I inhibitor (DXd) with a high drug-to-antibody ratio (DAR). In this study, the pharmacological activity of DS-3939a was evaluated using preclinical in vitro and in vivo models. Methods: TA-MUC1 expression was assessed by immunohistochemistry and flow cytometry. Binding specificity of DS-3939a was confirmed by enzyme-linked immunosorbent assay. Induction of DNA damage and apoptosis to cancer cells by DS-3939a treatment were detected by Simple Western System. In vitro cell growth inhibitory and in vivo antitumor activities of DS-3939a were evaluated using various cancer cell lines and patient-derived xenograft (PDX) models. Results: Many specimens across multiple cancer types showed positive staining for TA-MUC1, with a particularly high positive rate observed in bladder, lung, and breast cancers. DS-3939a specifically bound to aberrantly glycosylated MUC1 peptides by recognizing both the glycan and backbone peptide moiety of MUC1. In vitro, DS-3939a inhibited the growth of TA-MUC1-positive cancer cells but not of TA-MUC1-negative cancer cells. Neither a parental anti-TA-MUC1 antibody nor isotype control ADC inhibited the growth of these cells. In addition, treatment with DS-3939a and DXd alone induced DNA damage and apoptosis in cancer cells. These results demonstrate that DS-3939a exhibits TA-MUC1-dependent cell growth inhibition through DXd-mediated cytotoxic effect. Moreover, DS-3939a exhibited significant antitumor effects in the several TA-MUC1-positive cancer cell line-derived xenograft models and various PDX models. DS-3939a also induced strong tumor regression even after treatment of other cytotoxic ADCs in several xenograft models. Conclusion: Based on these preclinical results, DS-3939a could provide valuable therapy with potential benefits for patients with TA-MUC1-expressing tumors. A first-in-human phase 1/2 study in patients with advanced solid tumors is currently in progress (NCT05875168). Citation Format: Mayuko Yukiura, Kohei Takano, Kazuki Takahashi, Akiko Zembutsu, Michiko Kitamura, Yoshinobu Shiose, Kokichi Honda, Kazunori Oyama, Wataru Obuchi, Makiko Yamada, Riki Goto, Ken Sakurai, Kazuyoshi Kumagai, Takashi Kagari, Yuki Abe, Toshinori Agatsuma. DS-3939a, a novel TA-MUC1-targeting antibody-drug conjugate (ADC) with a DNA topoisomerase I inhibitor DXd, exhibits potent antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6579.

Abstract 5881: Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells

Abstract Ten-eleven-translocation 2 (TET2) is a member of the TET family proteins (TET1-3) that convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and causes site-specific DNA demethylation. The effects of DNMT1 on TET2 phenotype following DNMT inhibitor treatment is unclear in human cancer. In colorectal cancer HCT116 cells with DNMT1 gene intact (DNMT1+/+) and deletion (DNMT1−/−) status, we found that TET2 expression was robustly increased in DNMT1−/− cells after treatment with 0.5 µM and 5 µM decitabine for 72 h, 96 h, and 120 h. The augmentation in TET2 expression was accompanied by a dose-dependent re-expression of p16ink4A in DNMT1−/− cells. Particularly, TET2 upregulation was associated with resistance to DNMT inhibitors, showing a lack of induction of DNA damage, cell-cycle arrest and apoptosis after drug exposure in DNMT1−/− compared to DNMT1+/+ cells. The treatment with 4’-thio-5-aza-2’-deoxycytidine (aza-T-dCyd) only up-regulated TET2 and re-expressed p16ink4A at the lower dose of 0.5 µM, which was consistent with our previous findings that high dose of aza-T-dCyd at 5 µM predominantly eliminates the cancer cells with severe DNA damage by undergoing apoptosis and cell death (Scientific Reports, 13:5964, 2023). Moreover, both decitabine and aza-T-dCyd had minimal effects on TET2 upregulation and p16ink4A re-expression in cells with intact DNMT1 gene. The data suggest that the absence of DNMT1 gene made the cells prone to TET2 upregulation upon DNMT inhibitor challenges. Future study is needed to investigate the interactive relationship between DNMT1 and TET2 as well as demethylation, and its impact on cancer treatment with DNMT inhibitors. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Lorraine C. Pelosof, James H. Doroshow, Sherry X. Yang. Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5881.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 618.