CellTiter-Glo Viability Assay Research Articles

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 μg/mL of LPS, with or without 2 μM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 μg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 μg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

Abstract LB273: Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes

Abstract Introduction: Inhibition of poly(ADP-ribose) polymerase (PARP) induces synthetic lethality in cells exhibiting defects in homologous repair (HR) pathways, such as BRCA1 and BRCA2-mutated breast or ovarian cancers, and 4 different PARP inhibitors (PARPi) are currently approved by the FDA. While HR-deficient cancers usually respond well to single agent PARPi, HR-proficient cancers are generally less sensitive to the treatment. Acquired resistance to PARPi due to activating mutations in BRCA genes are also not uncommon. In this work, we have examined strategies for enhancing PARPi activity in a HR-proficient context and identified histone deacetylases (HDACs) as targets for pharmacological synthetic lethality in the context of PARPi. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 were determined using CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by immunoblotting and immunofluorescence using gamma-H2AX antibodies, and comet assays for confirmation of double-strand breaks. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module. In vivo metastasis formations were examined using the pulmonary metastasis assay (PuMA). Results and discussion: Combinations of PARP and HDAC inhibitors in HR-proficient tumor cells demonstrated that HDACi could enhance the efficacy of PARPi in vitro. This was associated with increased acute and sustained DNA-damage distinct cell cycle profiles showing activity in both S- and G2/M-phases. Importantly, dual single-molecule PARP-HDAC inhibitors showed potent inhibition of PARP1 and PARP2 activity and PAR synthesis, comparable to olaparib, as well as inhibition of HDAC with IC50 values in the low µM range. Dual PARP-HDAC inhibition decreased survival of HR-proficient cells in both 2D cultures and 3D spheroid assays with lower EC50 values than olaparib or vorinostat alone. Importantly, lung metastasis formation was significantly impaired by dual PARP-HDAC inhibition. Conclusion: Combination of PARP inhibition and HDAC inhibition showed increased efficacy compared to single agent PARP inhibition in HR-proficient cells. Development of bifunctional inhibitors may provide a novel therapeutic opportunity for tumors with limited response rates to single agent PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Fariba Ghaidi, Mona Marzban, Hans Adomat, Xiaoqi Chen, John Langlands, Dennis Brown, Jeffrey Bacha, Colin Collins, Poul H. Sorensen, Wang Shen, Mads Daugaard. Pharmacological synthetic lethality by co-inhibition of PARP and HDAC enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB273.

Abstract 3245: Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia

Abstract Large granular lymphocyte (LGL) leukemia is a rare heterogeneous blood cancer that is characterized by the clonal lymphoproliferation of CD8+ T cells (T-LGL leukemia) or, less frequently, natural killer cells (NK-LGL leukemia). These cells evade activation induced cell death, allowing them to accumulate as highly cytotoxic lymphocytes that contribute to autoimmune attacks, neutropenia, and anemia. Frontline treatments are limited to repurposed, nonspecific immunosuppressive agents with limited efficacy and incomplete response rates. As there are no FDA-approved antineoplastic agents for LGL leukemia, our research explores novel therapeutic avenues for this orphan disease. Somatic activating mutations in the transcription factor STAT3 are the most common molecular alteration found in >50% of patients, and STAT3 signaling is dysregulated in almost all LGL leukemia patients. In the largest molecularly profiled LGL leukemia patient cohort to date, we found that STAT3 mutations frequently co-occur with mutations in chromatin and epigenetic modifying genes, suggesting crosstalk and cooperativity between STAT3 activation and epigenetic dysregulation. Moreover, TET2, which promotes DNA demethylation, is frequently inactivated in LGL leukemia via loss-of-function mutations and promoter hypermethylation. As DNA hypermethylation is an emerging hallmark of LGL leukemia, we evaluated the therapeutic potential of two epigenetic agents, azacitidine (AZA) and cladribine (CLAD), that directly and indirectly inhibit DNA hypermethylation, respectively. We hypothesize that AZA and CLAD will 1) exhibit cell intrinsic efficacy, 2) reprogram the epigenome of LGL leukemia through DNA hypomethylation to normalize aberrant gene expression, and 3) induce cytokine-mediated immune responses. AZA and CLAD cytotoxicity was assessed by the Cell Titer Glo viability assay in human LGL leukemia cell lines. Each exhibited potent anti-leukemic effects with IC50 values in the low micromolar and nanomolar ranges. Western blotting analysis demonstrated the decrease in protein expression of DNA methyltransferases and phosphorylated STAT3 levels with treatment in a time- and dose-dependent manner. We also explored the immunomodulatory effects of these hypomethylating agents since LGL leukemia is a pro-inflammatory malignancy with cytotoxic lymphocytes. Preliminary findings using the Isoplexis platform suggest that AZA and CLAD induce production of anti-inflammatory cytokines in LGL leukemia cells. Ongoing studies will identify differential methylation, chromatin accessibility, and gene expression using RRBS, ATAC-seq, and RNA-seq of AZA or CLAD treated samples to define the mechanisms and consequences of DNA demethylation. Uncovering the reprogramming capabilities of AZA and CLAD could allow for the potential repurposing of these inhibitors for treatment of LGL leukemia patients. Citation Format: Ariana Sabzevari, Ipsita Pal, Tess Deddens, Todd Fox, Aakrosh Ratan, David J. Feith, Thomas P. Loughran. Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte 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 3245.

Genome-Wide CRISPR/Cas9 Knock-out Screen Identifies Apoptosis-Relevant Genes in Multiple Myeloma

Background Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β family and have several biological functions in different cells and tissues. In multiple myeloma, BMPs enable growth arrest and apoptosis of the cancer cells. BMPs act as ligands and signal by binding a tetrameric complex of type I and type II receptors, leading to activation of SMAD1/5/8 transcription factors which allocate into the cell nucleus. The main type I receptor in myeloma cells is ALK2, encoded by the ACVR1 gene. Several BMP ligands can signal via ALK2, but the downstream signaling and mechanisms leading to apoptosis and growth arrest remain unknown. Mapping of this cascade could result in potential novel targets for treatment and therapies. Methods A genome-wide CRISPR/Cas9 knock-out screen with the GeCKO v2 library was performed in INA-6 myeloma cells. To induce apoptosis and growth arrest two treatments were performed in parallel: BMP9 and a combination of Activin B and FK506 (tacrolimus), compared with medium control. Both BMP9 and Activin B signal via the type I receptor ALK2 combined with one or more of the type II receptors ACVR2A, ACVR2B or BMPR2. Top hits were defined as genes over- or underrepresented compared with control in both treatment conditions with a p-value lower than 0.05. Reactome pathway analysis of the top hits from the screen was performed. Validation of the top ranking hits was performed by generating single-gene specific knockouts, silencing with siRNA or with inhibitors. CellTiter Glo viability assay and annexin V/propidium iodide staining analyzed by flow cytometry were used to determine apoptosis. RT-PCR and western blots were used to determine specific mRNA and protein abundance. Results The screen identified genes already known to be necessary in the BMP signaling pathway, such as ACVR1 and SMAD1 indicating technical robustness. Although the screen was designed to pick genes necessary for apoptosis and growth arrest, we were also able to identify genes that counteract BMP-induced apoptosis. Several novel genes/hits were found in the screen. Numerous well-established signaling cascades were identified from Reactome pathway analysis to be activated downstream of BMP mediated signaling. In particular, several genes belonging to the PI3K/AKT/mTOR cascade were significantly overrepresented in the screen, indicating they positively regulate ALK2-mediated apoptosis. The PI3K/AKT/mTOR pathway is known to be upregulated in several cancers, including multiple myeloma. Transcriptional regulators of TP53 were found to be significantly underrepresented, indicating negative regulation in ALK2-mediated apoptosis. The protein encoded by the TP53 gene is well known from several malignancies, including multiple myeloma. Additionally, 9 other genes with previously unknown apoptosis function were found to be relevant for its regulation. Conclusion In conclusion, BMP-induced apoptosis in multiple myeloma cells rely on the canonical BMP signaling pathway, but also on a number of previously unknown genes. The screen confirmed the importance of the PI3K/AKT/mTOR pathway for positive apoptosis regulation and transcriptional factors affecting TP53 for negative apoptosis regulation. Hits from this screen can be promising targets for future therapies and precision medicine targeting the apoptotic machinery in multiple myeloma cells.

First-in-Class HAT Activator (YF2) Combined with JAK/STAT Inhibitor (ruxolitinib) Unveils Potential Novel Treatment Approach for HDAC Inhibitor-Resistant CTCL

T-cell lymphomas are both aggressive and challenging to treat. This is in-part due to multiple epigenetic derangements found in T-cell lymphomas. One treatment strategy includes HDAC inhibitors; however, the duration of response is short for most patients which is consistent with many treatments for T-cell lymphoma. As a result, alternative treatments are required. YF2, a first-in-class histone acetyltransferase (HAT) activator, was invented at Columbia University. YF2 increases acetylation of histones by augmenting the activity of HAT enzymes. The JAK/STAT pathway contributes to CTCL lymphomagenesis through a number of cellular processes including cell cycle, proliferation, apoptosis, etc. JAKs activate STAT3, which activates the transcription of the anti-apoptotic Bcl-2-like protein, Bcl-XL. Additionally, HATs acetylate the pro-apoptotic p53 protein, increasing its stability and activity. Given the dependence on both the JAK/STAT pathway and epigenetic derangements in CTCL, we hypothesize that if the JAK inhibitor, ruxolitinib, induces apoptosis via modulation of Bcl-XL, and YF2 induces apoptosis via acetylation and activation of p53, then dual targeting of these complementary pathways could lead to synergistic apoptosis in CTCL. To study this hypothesis, we used two CTCL cell lines: HH [JAK wildtype (JAK WT)], and H9 [JAK1/3 gain-of-function mutated (JAK1/3 GOF)]. In addition, we generated a belinostat-resistant H9 cell line (H9-belino-R) by incrementally exposing H9 to increasing concentrations of belinostat. H9-belino-R resistance was observed at a 20-fold (IC 50 = 896.7nM, SEM ± 185.3) increase over that of the parental H9 (IC 50 = 43.8nM, SEM ± 4.61) ( figure 1). H9-belino-R retained significant resistance to other HDAC inhibitors such as romidepsin [(H9: IC 50 = 0.97nM (SEM ± 0.030), H9-belino-R: IC 50 = 1.38nM (SEM ± 0.028)] and panobinostat [H9: IC 50 = 3.11nM (SEM ± 0.19), H9-belino-R: IC 50 = 9.00nM (SEM ± 1.55)] as measured by the CellTiter-Glo Viability Assay. Interestingly, the IC 50 of the HAT activator, YF2, was retained between the resistant (14.84µM, SEM ± 0.46) and parental (12.45µM, SEM ± 0.27) cell lines ( figure 1). HH (JAK WT) showed significant cytotoxicity (63.5% live, 30.7% apoptotic, and 5.4% dead) after 4-day exposure to 10µM YF2 compared to limited cytotoxicity (93.3% live, 4.6% apoptotic, and 2.0% dead) in H9 (JAK GOF) as measured by flow cytometry with Annexin V. At baseline, active STAT3 (pSTAT3) was not detected in HH possibly contributing to its susceptibility to YF2 as the absence of pSTAT3 reduces the transcription Bcl-XL. As illustrated by figure 2, western blot analysis confirmed ruxolitinib-induced inhibition of active STAT3 (pSTAT3) with associated reduction in Bcl-XL expression in H9 and H9-belino-R. This may explain the observed synergistic cytotoxicity with YF2 and ruxolitinib in HH, H9 and H9-belino-R (Excess over Bliss scores: 4.85, 14.23, and 5.38 respectively) as determined by flow cytometry. In conclusion, the novel first-in-class HAT activator, YF2, retains activity in HDAC inhibitor-resistant CTCL. YF2's activity may be blunted by JAK1/3 GOF mutation; however, when combined with the JAK-inhibitor ruxolitinib, the response mirrored that of the HH (JAK WT) cell line. The combination of HAT activator and JAK inhibitor led to enhanced reduction of the anti-apoptotic protein Bcl-XL, potentially explaining synergistic apoptosis in CTCL. These preliminary findings provide us with evidence that suggests that the combination of YF2 and ruxolitinib can serve as a novel treatment combination for CTCL. Further study is planned to explore this treatment in murine models of disease.

Abstract B068: Treatment combinations targeting MCL-1 and BCL-xL exert synergistic anti-tumor effects in AR-V7 expressing CRPC cell lines

Abstract Therapeutic agents that overcome acquired resistance to current androgen receptor (AR) pathway targeted therapies in patients with castration-resistant prostate cancer (CRPC) remain an unmet clinical need. A multitude of pathways are implicated in acquired treatment resistance, including the PI3K/mTOR/AKT, WNT/β-catenin, and DNA damage repair pathways. Notably, the androgen receptor splice variant 7 (AR-V7) activates the AR pathway in the absence of androgens, playing a crucial role in CRPC progression. An unbiased high throughput drug screen evaluating an array of inhibitors targeting diverse signaling pathways revealed highly synergistic activity of BH3 mimetics in AR-V7 expressing CRPC cell lines (e.g. LNCaP95, 22Rv1, VCaP-CR). Combinations targeting MCL-1 and BCL-xL (S63845 with A-1331852 or Navitoclax) displayed increased antitumor activity compared to those targeting MCL-1 and BCL-2 (S63845 with Venetoclax). Synergy among BCL-xL and MCL-1 targeted combinations was demonstrated using 2D and 3D CellTiter-Glo viability assays. Combination treatments in 3D spheroid cultures of 22RV1 and LNCaP95 cells confirmed the synergy observed in 2D assays. Furthermore, combined BCL-xL and MCL-1 inhibition altered AR-V7 expression and activated c-PARP cleavage. Drug combinations synergistically targeting BCL-xL and MCL-1 should be further explored for the treatment of therapy-resistant CRPC. Citation Format: Giulia C. Napoli, Andres F. Leon, Erica L. Beatson, Keith T. Schmidt, Emily N. Risdon, Cindy H. Chau, Douglas K. Price, William D. Figg. Treatment combinations targeting MCL-1 and BCL-xL exert synergistic anti-tumor effects in AR-V7 expressing CRPC cell lines [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B068.

Abstract 6194: A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models

Abstract Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.

Abstract 1066: PARP inhibitor rucaparib in combination with imipridones ONC201 or ONC212 demonstrates preclinical synergy against BRCA1/2-deficient breast, ovarian, and prostate cancer cells

Abstract BRCA1/2 genes encode proteins that mediate homologous recombination and repair (HRR). BRCA1/2 mutations increase risk of breast, ovarian, prostate and other cancers. BRCA1/2 mutated tumor cells are sensitive to PARP inhibitors (PARPi), that cause DNA replication fork to collapse. Approximately 40% of patients develop PARPi resistance through different mechanisms. One of the mechanisms of PARPi resistance is through P13k/Akt pathway activation. Imipridones are TRAIL-inducing compounds which are PERK-independent activators of the integrated stress response, and dual inhibitors of Akt/ERK. We hypothesized that combining imipridones with PARPi would overcome PARPi resistance due to Akt activation. PARPi also sensitize various solid tumors to recombinant TRAIL and DR5 agonist antibodies thereby further suggesting possible synergies between imipridones and PARPi. Our lab previously showed efficacy of imipridone ONC201 in BRCA-deficient cancer cells and potential synergy with olaparib (2017 AACR annual meeting abstract), without further investigating potential synergistic mechanisms. We explored combination drug synergy of rucaparib (PARP inhibitor) and imipridones (ONC212 and ONC201) in BRCA1/2-deficient breast, ovarian and prostate cancer cell lines. CellTiterGLO viability assays were performed after 72 hours to demonstrate synergistic effects of combination treatment. Western blots were performed to investigate the effect of combination treatment on the Akt pathway, as well as expression of cellular metabolic stress protein ATF4. Cytokine profiling using the Luminex 200 technology was used to study the effect of treatment on the tumor microenvironment. Combination treatment (rucaparib and ONC212, rucaparib and ONC201) in BRCA–deficient cell lines (HCC1937, PEO1, KURAMOCHI, 22RV1, LNCAP) showed synergistic reduction in cell viability. In the HCC 1937 cell line, combination studies of rucaparib-ONC212 and rucaparib-ONC201 showed a synergystic effect, as calculated by Compusyn software, combination indexes below one were observed at concentration of 0.29-37.5 of µM rucaparib with ONC201 at 1.25-5 µM and ONC212 6.25-50 nM with the best combination index of 0.7 for rucaparib-ONC201 combination and 0.31 for rucaparib-ONC212. We similarly observed synergy in other cell lines with combination treatment. Western blot analysis of rucaparib-ONC212 combination showed total Akt protein reduction and an increase in ATF4 consistent with the synergistic effects. Further studies are ongoing to characterize possible mechanisms and effects of PARP inhibitor-imipridone combination treatment on immune-mediated killing. Our findings identify novel PARP inhibitor-imipridone therapy combinations that can be further developed for treatment of BRCA1/2 deficient cancers. Citation Format: Maryam Ghandali, Kelsey E. Huntington, praveen Srinivasan, Don S. Dizon, Stephanie L. Graff, Benedito A. Carneiro, Wafik S. El-Deiry. PARP inhibitor rucaparib in combination with imipridones ONC201 or ONC212 demonstrates preclinical synergy against BRCA1/2-deficient breast, ovarian, and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1066.

Abstract 5512: TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant form of ovarian cancer. Although patients with HGSOC are likely to respond to treatment, it is common for the cancer to recur and become resistant to treatment, therefore there is a need to develop new targets and novel combinations to increase patient survival rates. ATRi’s (Ataxia telangiectasia and Rad3 related inhibitors) have shown efficacy in HGSOC. Considering ATRi’s have shown moderate efficacy as a single treatment in HGSOC patients, we hypothesized that combining ATRi’s with ONC201 would enhance efficacy. ONC201 is a pro-apoptotic TRAIL-inducing compound that activates the integrated stress response and inactivates the Akt pathway which is upregulated in over 70% of HGSOC. Inactivation of the Akt pathway may result in a synergistic effect that could be translated for use in patients with HGSOC. Three HGSOC cell lines were treated with the novel drug combination of ceralasertib (ATRi) and ONC201 and CellTiterGLO viability assays were performed after 72 hours to evaluate the synergistic effect of combination treatment. Western blots were performed to investigate the synergistic effect of combination treatment on the Akt pathway as well as expression of the cellular metabolic stress protein ATF4. Cytokine profiling using the Luminex 200 technology was performed to study treatment-induced changes in the tumor microenvironment. Cell viability after ceralasertib and ONC201 combination treatment showed synergy in HGSOC cell lines (OVCAR3, KURAMOCHI, TOV21G). In TOV21G cell line, combination studies of ONC201 and ceralasertib calculated by compusyn software demonstrated a synergistic effect of both drugs, Combination indexes below one were observed at concentration of 0.008-2 μM of ceralasertib with 0.31-5 μM of ONC201 with the best combination index of 0.65. We similarly observed synergy in the two other cell line between ONC201 and ceralasertib. Western blotting showed reduction in total Akt and Erk expression and an increase in ATF4, consistent with the observed synergetic effect of ceralasertib and ONC201 combination. Our ongoing studies are exploring potential mechanisms of synergy between ceralasertib and ONC201 and effects on immune–mediated killing of HGSOC cells. Our results suggest a rational and potentially effective novel therapy combination of ATRi ceralasertib and ONC201 that maybe further developed for HGSOC. Citation Format: Maryam Ghandali, Kadir Mert Selvi, Kelsey E. Huntington, Andrew George, Anna Ochsner, Benedito A. Carneiro, Donn S. Dizon, Wafik S. El-Deiry. TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5512.

Abstract 1070: The anti-tumor efficacy of combining oral ATR kinase inhibitor ceralasertib with TIC10/ONC201, an oral Akt/ERK inhibitor, TRAIL pathway and integrated stress response inducer, in prostate cancer treatment

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death among men in the United States. There are 268,490 estimated new cases and 34,500 estimated deaths in 2022 according to the NIH. Patients with metastatic castration resistant prostate cancer (mCRPC) have a poor prognosis and the current treatment options show limited efficacy. From the TRAP trial, 20% of mCRPC patients are reported to bear DNA repair defects. Ceralasertib, formerly known as AZD6738, is a potent and selective orally bioavailable inhibitor of the ataxia tenlangiectasia and Rad3-related (ATR) kinase, which is involved in DNA repair in response to DNA damage and replication stress. Ceralasertib’s antitumor activity as a monotherapy in treating prostate cancer is moderate. Thus, we investigated a combination therapy of ceralasertib with ONC201, a potent and cytotoxic orally bioavailable inhibitor of the serine/threonine protein kinase Akt and extracellular signal-regulated kinase (ERK). Upon administration, ONC201 can induce tumor cell apoptosis mediated by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and activate integrated stress response (IRS). Preliminary results demonstrate synergistic activity with the combination therapy in vitro using CellTiterGLO viability assay 72 hours in a 96 well plate post treatment. Ongoing studies using western blotting and cytokine profiling are intended to illustrate the mechanism behind the proven synergy. Our results aim to solve the overarching need in developing novel therapeutic strategies to overcome resistance in current prostate cancer treatments. Citation Format: Yutong Xia, Maximilian P. Schwermann, Andrew George, Anna Ochsner, Benedito A. Carneiro, Wafik S. El-Deiry. The anti-tumor efficacy of combining oral ATR kinase inhibitor ceralasertib with TIC10/ONC201, an oral Akt/ERK inhibitor, TRAIL pathway and integrated stress response inducer, in prostate cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1070.

Abstract 1822: IFN-γ and TRAIL involvement in androgen signaling-mediated NK cell killing of prostate cancer cells and further enhancement by NKG2A checkpoint inhibition with monalizumab

Abstract Although blocking the androgen receptor (AR) pathway is a core treatment strategy in early prostate cancer, most patients progress to metastatic castration-resistant prostate cancer(mCRPC). Immunotherapy with PD-1/PD-L1 inhibition has limited activity in mCRPC. AR signaling modulates CD8+ T cells and antitumor immune responses in mCRPC. In a companion presentation (Schwermann et al., submitted AACR 2023), we demonstrate that AR blockade activates NK cell killing of prostate cancer (PC) cells. We hypothesize this activation requires IFN-γ and TRAIL pathways. We investigated the mechanisms of AR inhibitors (ARi)enzalutamide (Enza) and darolutamide (Daro) on the antitumor function of NK cells and potential enhancement with checkpoint inhibitors. Material and Methods: We performed ATP-based CellTiterGlo viability assays to investigate the effects of Enza and Daro on PC cells (LNCap, 22Rv1 [ARv7mutation], DU145, PC3 [AR-]) and NK-92 cells. We performed co-culture experiments with PC, NK, and T cells, at a 1:1 ratio, using IFN-γ blocking mAb (10 μg/ml), RIK-2 (TRAIL blocking mAb- 10 μg/ml) and analyzed immune cell-mediated tumor cell killing (ImageXpress Confocal HT). The NK cell immune checkpoint inhibitor monalizumab that targets NKG2A was combined with ARi. Results: Co-cultures of PC plus NK-92 cells with ARi significantly increased immune-mediated PCkilling within 24hrs (control[C]: 20%±1.9; Enza: 40%±4.3; Daro: 36%±3.72, p=0.0001). This immune enhancement effect was abolished by IFN-γ mAb. The combination of TRAIL inhibitory mAb with Enza decreased NK immune-mediated killing by 16±2.4% ([C]: 40%±4.3; Enza+RIK-2:25.6%±2.1, p=0.002). Treatment of PC cells plus NK cells with Enza, IFN-γ mAb, and TRAIL-blocking RIK-2 significantly reduced NK cell-mediated killing of PCs (control[C]: 20%±1.9, [T]:3.2%±2.6, p=0.003). ARi immune enhancement effect was increased by NKG2A blockade with monalizumab ([C]: 31.2%±3.1, [T]: 43.92%±4.52, p=0.0045). Conclusions: ARi promotes NK cell killing of PC cells via IFN-γ gamma and TRAIL. NK cell activation is enhanced by the combination of ARi and the checkpoint inhibitor monalizumab. Validation of these results in patient-derived organoids paired with tumor samples from patients with PC is ongoing to further understand the innate immune response and direct novel therapeutic strategies. Citation Format: Maximilian Schwermann, Lindsey Carlsen, Kelsey E. Huntington, Lanlan Zhou, Andrew George, Praveen Srinivasan, Vida Tajiknia, Arielle De La Cruz, Andre De Souza, Anthony E. Mega, Howard P. Safran, Benedito A. Carneiro, Wafik S. El-Deiry. IFN-γ and TRAIL involvement in androgen signaling-mediated NK cell killing of prostate cancer cells and further enhancement by NKG2A checkpoint inhibition with monalizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1822.

9 Caged rucaparib: cancer directed delivery of PARPi

Poly (ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian cancer, improving progression free survival in the upfront and recurrent setting. Unfortunately, due to side effects patients often require dose reductions or altogether discontinue PARPi therapy. Additionally, due to bone marrow toxicity, PARPi cannot be effectively combined with chemotherapy. Recently, it was demonstrated that a ferrous iron-activated MEK inhibitor can circumvent MEKi related toxicity in non-tumorigenic tissues. Therefore, we developed a ferrous iron-activated form of rucaparib to target cancer cells while mitigating PARPi toxicity. Normal cells maintain a lower concentration of intracellular labile ferrous iron (Fe(II)) as compared with cancer cells. We introduced an Fe(II)-sensitive 1,2,4-trioxolane moiety at a reactive amine in rucaparib, creating a Fe(II)-activatable form of rucaparib (TRX-Ruc). A negative control, Di-Ruc, that cannot be activated by Fe(II), was also synthesized. Both compounds were then tested for their ability to inhibit PARP using a cell-free PARP1 assay. Cellular antiproliferative activity was tested in UWB –/– (BRCA1- deficient ovarian cancer cells) using the CellTiterGlo viability assay, and intracellular inhibition of PARP was evaluated with a parylation Western blot. In the cell free PARP1 inhibitor assay, the IC50 of rucaparib was 2.9 nM while TRX-Ruc had significantly decreased potency, with an IC50 of 92.3 nM. Di-Ruc had no inhibitory effect as expected. UWB –/– viability was decreased by rucaparib and TRX-Ruc with an equivalent IC50 of approximately 300 nM while Di-Ruc only had an effect at very high concentrations. To ensure the mechanism of TRX-Ruc on cell viability was due to PARP inhibition, a parylation western was performed. Both rucaparib and TRX-Ruc inhibited parylation while the negative control, Di-Ruc had only minimal effects. The addition of an Fe(II)-sensitive “TRX” moiety on rucaparib significantly reduces the biochemical activity of rucaparib in the absence of Fe(II). However, in an ovarian cancer cell line, the activity of TRX-ruc is restored leading to decreased cell viability, which we demonstrated was realized through the inhibition of PARP. This new Fe(II)-sensitive compound is an exciting and novel approach to deliver PAPRi specifically to cancer cells, potentially reducing toxicity and allowing effective combinations of PARPi with other cytotoxic therapies.

A Novel Antibody-Drug Conjugate Targeting CCR7 in Hematologic Malignancies

Introduction: CC-chemokine receptor 7 (CCR7) is expressed in lymphoid malignancies and is therefore an attractive target for antibody-drug conjugates (ADCs). JBH492 is a novel, CCR7-targeting ADC, comprised of a humanized anti-CCR7 IgG1 antibody, conjugated in a site-specific manner to the maytansinoid microtubule inhibitor DM4 via a cleavable linker. Methods: CCR7 expression was investigated in human and cynomolgus monkey normal tissue microarrays (TMA) by in situ hybridization (ISH). CCR7 expression across multiple malignancies was investigated by RNA sequencing (RNAseq). The cytotoxic ADC activity of JBH492 was evaluated in vitro in a CellTiter-Glo viability assay. To characterize the impact of the ADC on the CCL19/CCR7 axis, p-ERK was measured as a downstream signaling event of CCL19-mediated CCR7 pathway activation. The in vivo efficacy of JBH492 was investigated in multiple preclinical models of lymphoid malignancies, chosen to cover a range of various CCR7 expression levels, including a panel of 11 DLBCL patient derived xenograft (PDX) models. The safety of JBH492 was assessed in repeat dose toxicity studies conducted in cynomolgus monkeys. Results: RNAseq and flow cytometry reveal high CCR7 expression in lymphoid neoplasms, including chronic lymphocytic leukemia (CLL), B-cell and T-cell non-Hodgkin's lymphoma (NHL), with very limited expression in healthy tissues. Preclinical characterization of JBH492 demonstrates potent, target-dependent cytotoxic activity. In vitro binding studies support avidity-driven binding of JBH492 to CCR7-high tumor cells compared to CCR7-low healthy immune cells. Mechanistic studies of JBH492 suggest a dual mechanism of action, including payload-mediated cytotoxicity, and blockade of ligand-induced CCR7 signaling through antibody antagonistic functionality. In vivo studies showed robust and durable efficacy in multiple clinically relevant lymphoma models. JBH492 achieved PR or CR in 7/11 DLBCL PDX models (response rate ~64%). Nonclinical toxicology studies indicate a favorable safety profile with no notable changes in circulating immune cell populations. Conclusions: Our translational data support the hypothesis that JBH492 may have the potential to become a promising first-in-class ADC for the treatment of CCR7-expressing lymphoid malignancies. The first-in-human clinical trial in patients with refractory/relapsed CLL and non-Hodgkin's lymphoma is currently ongoing (NCT04240704).

Investigating the in vitro Antitumor Structure-activity Relationship of a Range of Cannabinolic Acid Derivatives

Aim: To investigate the in vitro structure-activity relationship (SAR) of a range of tetrahydrocannabinolic (THCA) and cannabidiolic (CBDA) derivatives using the PANC-1 tumor cell line (pancreas, ductal carcinoma). Materials and methods: The in vitro effects of a range of THCA and CBDA derivatives with different carbonyl group substituents were tested on the PANC-1 cells cell line using the CellTiter Glo Viability Assay (72 hours) and the XTT assay (48 hours). Results: A study of a series of THCA and CBDA derivatives containing different functional groups at the carbonyl nitrogen atom demonstrated that THCA amides have better inhibitory activity, on the PANC-1 tumor cell line, than CBDA derivatives. Conclusions: THCA derivatives have better inhibitory activity than CBDA analogs with the same substituents. It is noteworthy that even a slight change in the structure of the substituent of the amide or hydrazone moiety of the molecule has a dramatic effect on the activity of these compounds.

109 (PB099) - A novel dual PARP-HDAC inhibitor for treatment of Ewing sarcoma

Background: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), but clinical trials with olaparib failed to show significant clinical benefit. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs correlate with more malignant phenotypes in sarcomas and inhibition of HDAC in pre-clinical models of ES inhibits tumor growth. HDAC inhibition combined with PARP inhibition has promising results in vitro, however clinically, combination therapies often require complicated sequential dosing protocols due to different pharmacokinetic profiles and overlapping toxicities, that severely limits clinical utility. Here, we report the efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibition activity. Methods: PARP1 and PARP2 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit, the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and by PARylation assays. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. DNA damage was detected by western blot, immuno-fluorescence, and comet assays. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low μM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in increased DNA damage and S and G2/M cell cycle arrest as compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may offer a novel treatment opportunity for Ewing sarcoma and other solid tumors with limited responses to PARPi monotherapies. Conflict of interest: Board of Directors: J. Bacha and D. Brown are directors of Rakovina Therapeutics. Corporate-sponsored Research: S. Truong, B. Zhai, F. Ghaidi, L. Ramos, J. Joshi are employees of Rakovina Therapeutics. J. Langlands, M. Daugaard and W. Shen are consultants to Rakovina Therapeutics.

Abstract A024: In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma

Abstract Introduction: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), though clinical trials with olaparib failed to show significant clinical benefit. While single agent therapy proved inefficacious in the clinical treatment of ES, combination therapies may show anti-tumour activity. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs have been correlated to more malignant phenotypes in sarcomas and inhibition of HDAC in ES has been shown to be effective in inhibiting tumor growth. HDAC inhibition combined with PARP inhibition has been shown to sensitize cells to treatment in vitro, however clinically, combination therapies often require sequential administration due to different pharmacokinetic profiles and overlapping toxicities, severely limiting clinical utility. Here, we evaluate the activity and efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibiting activity. Methods: PARP1 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was measured using the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit. HDAC activity was measured using HeLa nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. Accumulation of phospho-histone H2AX (pH2AX) was detected by western blot using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low µM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in the increased accumulation of pH2AX by western blot and increased S and G2/M cell cycle arrest compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may result in a novel treatment opportunity for Ewing sarcoma. Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Poul Sorensen, Mads Daugaard. In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A024.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Successful Retreatment With Venetoclax in a Patient With Chronic Lymphocytic Leukemia.

Abstract 1063: Combination strategies to overcome doxorubicin induced senescence in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor-2 over-expression. Compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence. While immunotherapy, PARP inhibitors, and sacituzumab govitecan have shown benefit in a subset of TNBC patients, chemotherapy with doxorubicin remains a mainstay of treatment. Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells are able to secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. It has been suggested that senescence is a potential mechanism of resistance in doxorubicin (dox) treated cells. Transitioning senescence mediated resistance to apoptosis is crucial to the treatment of TNBC. The objective of this study was to evaluate the combination BCL-2 and HDAC inhibitors with dox to overcome resistance and induce apoptosis. Experimental Procedures: TNBC cell lines resistant and sensitive to doxorubicin were identified using a CellTiter-Glo Viability Assay and resistant cell lines were selected for further analysis. To assess proliferation, dox resistant TNBC cell lines were plated in 96-well plates. Cells were exposed to vehicle control, dox, BCL-2 inhibitor, HDAC inhibitor or the combination of dox and BCL-2 inhibitor or HDAC inhibitor for 72 hours. Cellular proliferation was assessed using the BioSpa live cell analysis system. Apoptosis at 24 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and as single agents. Immunoblotting was performed to evaluate the downstream effects of apoptosis and senescence of drugs as single agents and in combination. Results: The addition of a BCL-2 inhibitor and HDAC inhibitor to doxorubicin resulted in decreased proliferation in resistant TNBC cell lines compared to single agents and vehicle control by live cell microscopy. Furthermore, the combination of dox and a BCL-2 inhibitor resulted in increased apoptosis when compared to single agents and control using Annexin V staining. The cyclin dependent kinase inhibitors p21 and p16 were over-expressed in cells exposed to combination treatment. Apoptotic proteins BAD, PUMA, cleaved PARP and the anti-apoptotic protein BCL-2 were upregulated in cells treated with a BCL-2 inhibitor with or without dox. The pro-mitotic protein cyclin-B1 was downregulated in resistant cells treated with single agent BCL-2 inhibitor and in combination with dox. Additional mechanistic studies are ongoing. Conclusion: The combination of doxorubicin with BCL-2 and HDAC inhibitors resulted in decreased cellular proliferation and increased apoptosis. Potential senolytic drugs used with dox represent an exciting potential to overcome dox resistance and warrant further investigation. Citation Format: Anna R. Schreiber, Stephen Smoots, Betelehem W. Yacob, Adrian TA Dominguez, Cecilia Levandowski, Jennifer R. Diamond, Todd M. Pitts. Combination strategies to overcome doxorubicin induced senescence in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1063.

Abstract 2661: Novel pharmacological approaches of inhibiting the PI3K/AKT/mTOR signaling pathway in T-cell lymphoma (TCL)

Abstract Introduction: T-cell lymphoma (TCL) accounts for 15% of all non-Hodgkin’s lymphoma cases with most patients developing relapsed/refractory (R/R) disease. The management of R/R TCL is challenging and responses to second and beyond-line therapy are generally poor. Pre-clinical and clinical studies have identified the relevance of PI3K/AKT/mTOR signaling pathway in TCL biology. The combination of PI3K inhibitor (PI3Ki) Duvelisib and the HDACi Romidepsin improved the overall response rate (ORR) of R/R TCL to 50-55% in a phase I/II study (Horwitz et. al. ASH 2018, Lugano 2021). Evaluation of PI3Ki and HDACi combinations in TCL pre-clinical models is needed to guide the design of future clinical trials. Methods: A panel of TCL cells lines were chosen representing T-cell acute lymphoblastic leukemia, T-lymphoblastic lymphoma, cutaneous T-cell lymphoma. A panel of PI3Ki (Omipalisib, Umbralisib and Duvelisib) and HDACi (Entinostat and Romidepsin) were selected to determine which combination would be most synergistic. Entinostat, an oral HDACi, was chosen for its remarkable preclinical and clinical activity in other lymphoma subtypes. Umbralisib is a new PI3Kδ inhibitor which was recently approved for treatment of follicular lymphoma and marginal zone lymphoma. Cells were exposed to Omipalisib (3.125-100nM), Umbralisib (1-320uM), or Duvelisib (1-100uM) alone or in combination with Entinostat (0.08-10uM) or Romidepsin (0.25-6) for 24, 48 and 72 hrs. Cell viability was measured using the CellTiter-Glo viability assay. Half-maximal inhibitory concentration (IC50) at 72hrs was determined for each drug and coefficient of synergy was calculated using Calcusyn Software. Changes in cellular metabolism and cell cycle distribution after exposure to PI3Ki were determined using DiOC6 staining and Propidium Iodide staining respectively. Epigenetic changes following exposure to Entinostat or Romidepsin were investigated for possible mechanisms. Results: In vitro exposure of TCL cell lines to PI3Ki or HDACi resulted in dose-dependent cell death. The IC50 at 72 hrs was 30-500nM for Omipalisib, 9-35uM for Umbralisib, 1-35uM for Duvelisib, 0.2-2uM for Entinostat and for 1-4nM Romidepsin. All HDACi and Pi3Ki combinations demonstrated additive and/or synergistic activity. Duvelisib combined with Entinostat or Romidepsin exhibited the most synergy. Methylation profiling demonstrated genomic wide hypomethylation, specifically DNMT3A, in Romidepsin treated samples compared to Entinostat. Conclusions: PI3Ki and HDACi combinations show significant synergy in TCL preclinical models. DNMT3A hypomethylation following Romidepsin exposure is being explored as the basis of PI3Ki and HDACi synergy. If confirmed in clinical trials, these results may refine the tolerability of the regimen for patients treated with this very promising PI3Ki/HDACi combination. Citation Format: Gabrielle Hartman, Cory Mavis, Juan J. Gu, Amber White, Taylor Mandeville, Pallawi Torka, Paola Ghione, Francisco J. Hernandez-Ilizaliturri. Novel pharmacological approaches of inhibiting the PI3K/AKT/mTOR signaling pathway in T-cell lymphoma (TCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2661.

Excessive new origin firing underlies selective glioma stem cell cytotoxicity induced by replication stress response inhibition

Abstract Aims Glioblastoma (GBM) is a treatment refractory cancer of extreme unmet need which exhibits treatment resistance due to a subpopulation of GBM cancer stem cells which have constitutive DNA damage response activation driven by elevated replication stress (RS). RS response inhibition is potently cytotoxic to GSC, however mechanistic understanding will be key to biomarker discovery and successful clinical translation. We investigated response to combined ATR and PARP inhibition (CAiPi) to gain mechanistic insight and inform biomarker development. Method A panel of patient-derived GBM cell lines were cultured as stem enriched (GSCs) or stem depleted (bulk), to characterise response to combined ATR inhibition (VE821 5μM) and PARP inhibition (Olaparib 1μM), by CellTiter-Glo viability assay. Mechanistic investigations included immunofluorescence of 53BP1 nuclear bodies and DNA fibre analysis. Studies into the importance of PARP trapping included another PARPi Veliparib (1μM), and investigations into inhibition of origin firing used the CDK inhibitor Roscovitine. Results Responses to CAiPi in a panel of primary paired GBM GSCs vs differentiated progeny were heterogenous. CAiPi is selectively GSC cytotoxic in a subpopulation of tumours. DNA fibre analysis identified increased new origin firing with PARPi, which was correlated with increased PARP trapping. Inhibition of origin firing by exposure to roscovitine rescued the CAiPi cytotoxic phenotype, suggesting origin firing has an important role in selective GSC cytotoxicity. A population of treatment-sensitive GSCs with increased numbers of 53BP1 nuclear bodies in G1 phase with CAiPi were identified, indicative of under-replication of DNA in S phase. Conclusion Selective GSC cytotoxicity is induced by CAiPi via dysregulation of replication, by both DNA under-replication resulting in DNA lesions, and the novel finding of increased new origin firing in GSC due to PARPi.