Abstract 3167: Longer and brighter live cell bioluminescence prosubstrates

CT7439 is a small molecule kinase inhibitor of cyclin-dependent kinases CDK12/CDK13 that received FDA IND approval in 2023. In addition to inhibiting CDK12/13, CT7439 acts as a monovalent glue degrader of cyclin K (CCNK), the cyclin required for CDK12/13 function. Proteomics quantification by DIA LCS/LCSMS mass spectrometry was conducted after exposure to 30 nM CT7439 at 4 & 24 hr. OV-90 cells were cultured in media and exposed to CT7439 at various concentrations and time points. Combination studies were conducted with the PARP inhibitors and Carboplatin. The following parameters were assessed: proliferation/cell survival, confluency formation, CCNK protein levels and DNA damage and repair (DDR) genes. CT7439 inhibited OV-90 cell growth with an IC50 of 4.4nM and this impact on cell growth was significantly attenuated by co-treatment of the cells with proteasome inhibitor. Proteomics analysis revealed that 99 proteins were found to be differentially abundant between samples from CT7439 (30 nM) treated and vehicle at 4 hrs. At 24 hrs 1, 666 proteins were found to be differentially abundant. CCNK was the only cyclin impacted by CT7439. Pathway and ontology analysis showed reduced abundance for proteins involved in DDR. DDR genes (BRCA1, ERCC4, RAD51C and POLQ) are inhibited by treatment with CT7439, at concentrations consistent with inhibition of CDK12/13-Cyclin K in the cells. CT7439 activity in OV-90 in cellular assays: Data is mean ± SD (**Western blot, *qPCR) In addition to single agent activity of CT7439 a strong combination activity between CT7439 and the PARP inhibitors Olaparib, Niraparib and AZD10530 as well as the carboplatin was observed in an OV-90 cellular confluency assay. CT7439 is an orally bioavailable selective CDK12/13 inhibitor and degrader of CCNK. Pre-clinical studies demonstrates clinical potential for monotherapy use in solid tumors and as a combination therapy with agents known to cause a DNA damage response. Citation Format: Ashwani K. Bahl, Matthew Carr, Stuart Thompson, Adrian Campbell, Andrew Leishman, Edward Ainscow. CT7439 an oral first-in class selective CDK12 and 13 inhibitor and cyclin K degrader: Profiling activity and combination efficacy in an ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7004.

Dysregulation of protein-protein interactions between the tumor suppressor p53 and its binding partners is implicated in the pathogenesis of various cancers. Here we describe several novel assays for analysis of p53 interactions and their inhibitors both in live mammalian cells and in vitro. To evaluate putative inhibitors of protein-protein interactions between p53 and its negative regulators Mdm2 and Mdm4, we recently developed two comparative live-cell Fluorescent-Two Hybrid (F2H) assays. The F2H principle is based on a tethering strategy: the GFP-tagged protein (here p53) is enriched at the protein interaction platform of the engineered F2H-BHK cells and serves as bait, whereas the RFP-tagged protein serves as a prey (here Mdm2 or Mdm4). By performing p53:Mdm2 and p53:Mdm4 F2H assays side-by-side, we could evaluate the dual inhibitory activity of the previously published stapled peptides. Furthermore, since F2H allows visualization of the dynamics of protein-protein interactions, we could compare the compound's kinetics with real-time imaging. We performed a mutant analysis with F2H and showed that several Nutlin-resistant mutants of Mdm2 are sensitive to inhibition with stapled peptides sMTide-02 and sMTide-02a. For in vitro validation of p53 interactions, we developed novel p53 immunoprecipitation reagents. We employed the single-domain antibody technology in conjunction with phage display to isolate two specific anti-p53 VHHs (also termed nanobodies) from immunized alpacas. When conjugated to agarose beads, these VHHs serve as highly efficient pull-down reagents (p53-Traps), specific exclusively against N- and C-terminus of p53 respectively. Using both p53-Traps, we could confirm our F2H results in immunoprecipitation followed by Western blotting. Furthermore, p53-Traps enabled us to extend our analysis of the p53 interaction network and evaluate interactions between the p53 isoforms. Taken together, we developed a toolbox for analysis of p53 interactions both biochemically and by fluorescence microscopy. Our fully reversible live-cell F2H assays can be applied for side-by-side profiling of new inhibitors of p53:Mdm2 and p53:Mdm4 interactions with respect to their intracellular activity, cell penetration and kinetics. Our N- and C-terminal p53-Traps complement p53 interaction analysis and enable highly efficient biochemical investigation of the p53 network and discovery of new interaction partners of p53. Citation Format: Larisa Yurlova, Andrea Buchfellner, Benjamin Ruf, Sebastien Gabriel Michel Jo, Jean-Christophe Bourdon, Farid J. Ghadessy, Christopher J. Brown, David P. Lane, Tina Romer. Live cell and in vitro analysis of p53 interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1231. doi:10.1158/1538-7445.AM2015-1231

When studying the complexities of cancer biomarkers, the ability to monitor gene expression within live cells that have become cancerous provides better insight than examining fixed or lysed tissues. However, monitoring RNA within intact cells can prove to be challenging with current techniques, which involve complex RNA isolation procedures that compromise cell viability. Further, amplification methods can create false positives or erroneously inflate differences. Here we describe a probe-based detection method that allows for the detection of ErbB-2 and EGFR mRNA levels across live intact SK-BR-3 breast cancer cells without the need for transfection reagents. The detection methodology is based upon an oligonucleotide gold nanoparticle conjugate capable of detecting intracellular levels of mRNA and miRNA in living cells. The probes enter the cell by means of the cell's native endocytosis machinery. Upon binding to the complementary target RNA sequence, a fluorescent signal (flare) is released, which allows the cells to be detected on any fluorescence analysis platform. In contrast to traditional RNA detection methods, since our technique allows for detection within live cells without altering their gene expression, the same cells that were profiled for EGFR and ErbB-2 expression could be sorted based on their specific mRNA content and subsequently further studied. Stimulation or treatments could then be performed on the previously profiled cells and assessed using probes for additional RNA markers or more traditional techniques such as antibody staining, qRT-PCR, or Array profiling. For this purpose, we combined immunostaining for cell surface EGFR with fluorogenic nanoparticle-based detection of EGFR mRNA in 4 breast cancer cell lines known to range in EGFR expression levels, and observed significant correlation for the signals in two-color flow cytometry. Thus, detecting RNA expression levels in live cancer cells with the ability to utilize the same cells in downstream testing gives researchers the ability to perform experiments which were previously thought to be impossible. Citation Format: Don Weldon, Kevin Su, Alex Ko, Grace Johnston, Matthew Hsu, Lucas Armstrong. A novel approach to detecting RNA expression in living breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2013-LB-240

Breast cancer is a serious disease that affects millions of women worldwide, and 700,000 people succumb to the disease annually. Breast cancers are divided into several functional subgroups based on molecular characteristics and growth pattern: Luminal A and B, HER2 positive (HER2+) and triple negative. The subgroups are important from a clinical perspective as they partially guide treatment strategies. In this study we wanted to investigate the difference in target engagement with four drugs indicated for treatment of different types of breast cancer: Alpelisib, Neratinib, Vinorelbine, and Docetaxel. Five different human breast cancer cell lines were included, representing Luminal A (MCF-7, T-47D), HER2+ (SK-BR-3, BT-474), and triple negative (BT-20). We used CETSA® (CEllular Thermal Shift Assay) coupled to mass spectrometry detection to assess proteome wide changes in protein thermal stability in intact breast cancer cell lines. CETSA is unique as a target engagement detection modality as it allows for target engagement assessment in intact cells with unaltered drug molecules. Also, as biology is intact in live cells it is possible to follow compound induced downstream signaling or phenotypic events that result in changed thermal stability of proteins. In addition to assessing protein thermal stability with CETSA, which is a short treatment of 1 h, we also subjected cells in culture to prolonged drug exposures, ≤72 h, in order to measure viability. This was done to correlate early changes in protein thermal stability to the viability phenotype that manifested later. In general, responses both in protein thermal stability and cell viability was heterogenous among the cell lines. The most homogenous response in viability was observed for the Phosphatidyl Inositol 3’ kinase inhibitor Alpelisib, which reduced viability to 20 - 40% in all cell lines. In contrast, partial resistance was shown to the other drugs by at least one of the cell lines. For Alpelisib, the amount and type of proteins that were thermally shifted correlated with the effect on viability. The covalent HER2 inhibitor Neratinib was most effective in inhibiting viability in HER2+ cells and proteins of the HER2 pathway were thermally shifted in these cells, which was not observed for the other cell lines. Viability of the other cell lines were also affected by Neratinib, but only at higher concentrations. Both microtubule inhibitors induced thermal stabilization of tubulins, but that did not always translate into efficacy in the viability assay. However, sensitive cell lines showed thermal shifts of tubulins at lower concentrations of compound. In oncology drug development it is important that effects on viability are driven by on-target efficacy and not by off-target effects. Here we show the benefit of connecting knowledge of target engagement and phenotypic readouts; what targets are engaged, at which concentrations, and what is the corresponding phenotype. Citation Format: Tomas Friman, Tuomas Tolvanen, Merve Kacal, Victoria Brehmer, Stina Lundgren, Laurence Arnold, Daniel Martinez Molina. Target engagement sheds light on difference in drug efficacy in breast cancer cell lines [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 4723.

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging

Tumors with defects in mismatch repair genes can present with microsatellite instability (MSI) characterized by expansions of short, repeated DNA sequences. Functional genomic screens revealed that MSI-high (MSI-H) cell lines were uniquely vulnerable to depletion of Werner (WRN) which encodes an ATP-dependent helicase. Importantly inhibitors of WRN phenocopy genetic depletion leading to selective inhibition of MSI-H tumor growth. SNV5686 is a novel, reversible inhibitor of WRN with potent inhibitory activity in biochemical and cellular assays. SNV5686 shows exquisite selectivity against related RECQ helicases. The viability of MSI-H cells is selectively inhibited by SNV5686 through a mechanism involving activation of the DNA Damage response, loss of WRN protein and time- and dose-dependent increases in DNA breaks. In vivo, oral administration of low daily doses of SNV5686 potently depletes WRN and induces p21 in tumors and leads to deep tumor regression in an MSI-H colorectal xenograft model. Irreversible WRN inhibitors and the non-covalent inhibitor HRO761 leverage binding to CYS727 in the active site to attain potency and selectivity, and published data show that mutation of CYS727 severely abrogates their activity. SNV5686 in contrast retains potent inhibition of WRN-C727S in both biochemical and cellular assays thereby exhibiting a differentiated profile. Acquired resistance to covalent inhibitors against other cancer targets such as EGFR and BTK though mutation of the covalent-bonded CYS (EGFR-C797, BTK-C481) is a well-documented clinical phenomenon. By maintaining potency SNV5686 has the potential to be a best-in-class inhibitor of WRN through its activity against both WT and mutant WRN as well as favorable physical-chemical properties and excellent preclinical PK profile. Furthermore, toxicology studies with SNV5686 have not revealed adverse findings in hematology or clinical chemistry supporting the potential to combine with standard of care agents including chemotherapy. These data support the clinical investigation of SNV5686 for tumors characterized by MSI-H. Citation Format: Phillip C.C. Liu, Jeffrey Yang, Daniele Ferrante, Lin You, Mingming Gao, Yu Bai, Qipeng Fang, Yu Li, Zhentian Li, Chunhong He, Liyang Wang, Fei Zhou, Robert Landman, Hewen Zheng, Ken Mukai, Sabiha Chowdhury, Colin Zhang, Chao Qi, Liangxing Wu, Wenqing Yao. SNV5686 a differentiated inhibitor of Werner helicase with potent activity against both WT WRN and WRN-C727S mutant proteins for treatment of MSI-H tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2921.

Introduction: Immune checkpoint blocking antibodies achieved great clinical success, yet a large fraction of cancer patients receive minimum benefit from current immunotherapies targeted PD-1 and CTLA-4. As a new multifunctional immune checkpoint molecule, TIGIT holds promise to become an important cancer immune therapy target. AK127 is a novel humanized immunoglobulin (Ig) G1 monoclonal antibody (mAb) targeting T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT). AK127 binds to TIGIT specifically and blocks the interaction of TIGIT with its ligands, thus relieves immune suppression and promotes anti-tumor immune response. Method: The effect of AK127 on secretion of IL-2 in mixed culture of Jurkat-TIGIT and HT1080-aCD3scFv cells (ELISA) was determined in cellular assays. Binding kinetics of AK127 to C1q, FcγR were measured by Fortebio. ADCC, ADCP and CDC activities were determined in cellular assays. In preclinical in-vivo pharmacology studies, synergistic anti-tumor activity of AK127 and a bi-specific anti-PD1/CTLA4 antibody Cadonilimab (AK104) was investigated in BALB/c-hPD-1/hTIGIT transgenic mice with CT26.WT tumor model. Result: AK127 blocked the immune inhibitory signal mediated by TIGIT, resulting in enhanced IL-2 secretion. AK127 exhibited binding to FcγIa, FcγRIIIa and C1q, and eliciting ADCC, ADCP and CDC. In animal models, both AK104 (0.5 mg/kg) and AK127 (4 mg/kg) significantly inhibited tumor growth. The combination of AK127 and AK104 produced a significantly enhanced anti-tumor effect than either monotherapy, demonstrating the synergistic anti-tumor activity of AK127 combined with AK104. Conclusion: AK127 binds to human TIGIT with high affinity, competitively blocks the binding of TIGIT with its ligand CD155 and CD112, relieves immune suppression in both cellular and animal model systems, and promotes anti-tumor immune response. Citation Format: Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Dennis Xia, Peng Zhang, Max Wang, Michelle Xia, Baiyong Li. AK127, a novel monoclonal antibody (mAb) targeting T cell immunoreceptor (TIGIT) [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 5524.

The aim of this project is to understand the various mechanisms contributing to drug resistance development in cancer therapy. The efficacy of therapeutics is dependent on a drug binding to its target. We have developed a method that allows for the first time to directly evaluate drug binding to target proteins in cells and tissue samples the cellular thermal shift assay (CETSA) (Martinez Molina et al. Science, 341:84). CETSA is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. By monitoring the drug occupancy in the target protein, CETSA can be used to study processes of drug transport and metabolism in cancer cells. We have used CETSA to study the acquired drug resistance of, antifolate and fluropyrimidine drugs in pairs of parental and resistant cell lines. CETSA shifts and isothermal dose response fingerprint (ITDRF) were used to study the relative drug target engagement in these cells. Quantitative mass spectrometry was used to monitor differences in protein expression levels across the cell lines. Based on the CETSA measurements, resistant cells clearly showed a higher drug dose threshold as compared to the parent cell lines, typically requiring 8-50 times higher dose to establish similar target engagement. Several potential mechanism for drug resistant emerged - we, for example, observed up-regulation of thymidylate synthase and down regulation of reduced folate carrier (RFC) protein associated with antifolate transport, in some resistant cell lines. The data supports that CETSA is a potential valuable tool to dissect various mechanisms those contribute to resistant development in cancer cells. Citation Format: Lekshmy Kunjamma Usha Sreekumar, Yan Ting Lim, Saranya Veerappan, Par Nordlund. Exploring the potential of cellular thermal shift assay (CETSA) to study drug resistance during cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2017-2045

Chemoresistance often develops during drug treatment in cancer patients but its mechanism remains unclear. To understand acquisition of drug resistance, we investigated whether tumor cells dying during drug treatment might signal to neighboring live cells to program themselves for survival in the drug-containing environment. The experimental design was to identify a factor in the supernatants of docetaxel (DTX)-treated human DU145 and PC3 prostate tumor cells that could cause live tumor cells to resist apoptosis and cell death under docetaxel. We discovered that dying cells release HMGB1 which can bind TLR4 or RAGE on live tumor cells causing the induction of cytoplasmic Clusterin (CLU). We demonstrated that CLU is a potent prosurvival protein that can trap Bax from translocating to the mitochondria to cause caspade 3 activation and subsequent apoptosis. Proof of this pathway came from the ability of anti-HMGB1to prevent supernatants from dying cells to induce CLU in newly-plated tumor cells. The receptors for HMGB1 were identified by use of anti-TLR4 and anti-RAGE, which prevented recombinant HMGB1 or dying cell supernatants from inducing CLU in newly-plated tumor cells, causing them to subsequently succumb to DTX toxicity. Moreover, overexpression of anti-sense CLU in tumor cells abrogated their ability to respond to recombinant HMGB1 or dying cell supernatants to induce drug resistance. Most importantly, standard chemotherapeutic agents including gemcitabine, taxol, Ara-C, doxorubicin, cisplatin, etoposide and carboplatin, can cause the release of HMGB1 from dying tumor cells, implicating this crosstalk between dying and live tumor cells as a common pathway towards multi-drug resistance. Citation Format: Junmin Zhou, Xianghong Chen, Danielle L. Gilvary, Melba Marie Tejera, Erika A. Eksioglu, Thu Le Trinh, Sheng Wei, Julie Djeu. Acquisition of chemoresistance in tumor cells requires crosstalk between dying and remnant live tumor cells via HMGB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 875.

Introduction: Soft tissue sarcomas (STS) are heterogeneous mesenchymal tumors which are both morbid and lethal. G100 is a stable oil-in-water emulsion of glucopyranosyl lipid adjuvant, a highly potent toll-like receptor 4 (TLR4) agonist, which has been utilized for intratumoral (IT) injections and as vaccine adjuvants without significant toxicity. We hypothesized that IT G100 would induce a robust local and potentially systemic anti-tumor immune response in the STS microenvironment, leading to improved outcomes. Methods: 15 metastatic STS patients who had a superficial injectable lesion were treated with weekly IT G100 for 8-12 weeks; 12 patients received concurrent radiation for 2 weeks at the start, while 3 got IT G100 alone for 6 weeks prior to radiation. Biopsies and blood were collected pre and post treatment, and flow cytometry was performed on fresh tumor samples. T-cell receptor (TCR) deep sequencing of tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) was performed on 7 patients. RECIST v1.1 and the Common Terminology Criteria for Adverse Events were used to monitor clinical outcomes. Results: Patients had a median of 3 (0~5) prior lines of therapy and mean tumor size of 5.6cm (1~20cm). No grade 3 or higher treatment-related toxicity was observed, and local tumor control was achieved in 93% (14/15). 6 (40%) had stable disease after treatment, and 1 (P06) had complete regression of injected tumor. This tumor had a high percentage of infiltrating pre-treatment immune cells (12% CD45+ on flow cytometry versus 2.7% for all other tumors). TCR sequencing showed that the increase in clonality of PBMC after treatment was greater in P06 (389%) compared to 6 other patients (mean 34%). There was also higher overlap in TCR sequence between TIL versus PBMC after treatment (13% versus 22%), suggesting systemic expansion of tumor-specific T-cells. In 7 patients evaluable for tumor-associated macrophages (tumors with >1000 CD45+CD11b+ cells), 71% had a shift from an M2 to M1 phenotype. In all patients who received G100 alone, there was an increase in T-cell infiltration into tumor after treatment. In one patient (P14), the proportion of CD3+ live cells in tumor went from <1% to 62%; of these, 51% were CD4+ and 44% were CD8+. TIL from P14 had a 251% increase in clonality after treatment; at the same time, percentage of live tumor cells that were PD-L1+ increased from 0.02% to 1.3%. Conclusion: IT G100 provides a potentially viable agent for local control of metastatic STS. With or without radiation, G100 appears to shift the tumor microenvironment into a more inflammatory state with significant infiltration of T cells. The increase in clonality in PBMC and TIL, as well as increased overlap of tumor-associated versus peripheral TCR sequences, suggest induction of a tumor-specific response. Combination of G100 with other immunomodulators may further enhance the adaptive anti-tumor response. Citation Format: Yongwoo D. Seo, Edward Y. Kim, Ernest U. Conrad, Ryan B. O'Malley, Sara Cooper, Bailey Donahue, Lee D. Cranmer, Hailing Lu, Frank Hsu, Elizabeth T. Loggers, Taylor Hain, Darin J. Davidson, Lynn Bonham, Venu G. Pillarisetty, Gabrielle M. Kane, Stanley R. Riddell, Robin L. Jones, Seth M. Pollack. Intratumoral injection of the toll-like receptor 4 agonist G100 induces a T-cell response in the soft tissue sarcoma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2947. doi:10.1158/1538-7445.AM2017-2947

Context: Slits, representative axon guidance molecules, and their Roundabout (Robo) transmembrane receptors are identified as key regulators of many cancers. One genome-wide liknage analysis suggested Slit-Robo Rho GTPase activating protein 1 gene as a candidate gene in papillary thyroid carcinoma (PTC) susceptibility. Objective: To identify the role of Slit-Robo signaling in PTC. Results : Slits(Slit 1-3) and Robos (Robo 1-4) were expressed in all of thyroid cancer cells and Slit2, Slit3, Robo1 and Robo4 presented higher expression than others. The mRNA expression of Slits and Robos was higher in fresh frozen PTC tissues than matched normal tissues. When we conducted immunohistochemical staining of Slit2 and Robo1 in thyroid tissues, the positivity of Slit2 and Robo1 were higher in PTC tissues than benign or normal tissues. However, negative Slit2 expression was significantly associated with cervical lymph node (LN) metastasis, distant metastasis and cervical recurrence of PTC (P <.001) irrespective of BRAF mutation status. In cellular assays, Slit2 treatment inhibited proliferation of thyroid cancer cells as dose dependent manner and also significantly suppressed cell migration by inhibiting activation of beta-catenin and inducing E-cadherin. Slit2 treatment also significantly suppressed beta-catenin activation induced by Wnt3A in luciferase reporter assay. Conclusions : Low expression of Slit2 was associated with poor prognostic factors in PTC. This present study demonstrated the potential of Slit2 as a novel prognostic and therapeutic factor in PTC. Citation Format: MinJi Jeon, Won Gu Kim, Seonhee Lim, Eun Kyung Jang, Tae Yong Kim, Young Kee Shong, Won Bae Kim. Low Slit2 expression is associated with the aggressiveness of papillary thyroid carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2015-2042

BTK, an essential component of the BCR pathway, has emerged as novel target in the treatment of B-cell malignancies. Although Ibrutinib, the first-in-class irreversible inhibitor of BTK, showed promising clinical activity, recent study revealed that ibrutinib could antagonize rituximab induced antigen-dependent cell-mediated cytotoxicity (ADCC) by inhibiting ITK kinase activity (Kohrt et. al., 2013), suggesting the potential limitations in its clinical application. BGB-3111 is a novel, highly selective, second generation BTK inhibitor, currently under clinical investigation in hematological cancers. Its biochemical, cellular and in vivo activities were reported in this study. In both biochemical and cellular assays, BGB-3111 demonstrated nanomolar BTK inhibition activity. In several MCL and DLBCL cell lines, BGB-3111 inhibited BCR aggregation-triggered BTK autophosphorylation, blocked downstream PLC-γ2 signaling, and potently inhibited cell proliferation. In comparison with ibrutinib, BGB-3111 showed much more restricted off-target activities against a panel of kinases, including ITK. While ibrutinib significantly inhibited rituximab-induced NK cell IFN-γ secretion and in vitro cytotoxicity on mantle cell lymphoma cells, BGB-3111 was at least 10-fold weaker than ibrutinib in inhibiting rituximab induced ADCC, consistent with its weak ITK inhibition activity. In mouse BTK occupancy assays, treatment with BGB-3111 resulted in a dose-dependent BTK occupancy and showed about 3-fold more potency than ibrutinib in target organs, including PBMC and spleen. BGB-3111 induced dose-dependent anti-tumor effects against REC-1 MCL xenografts engrafted either subcutaneously or systemically via tail vein injection in mice. In the subcutaneous xenografts, BGB-3111 at 2.5 mg/kg BID showed similar activity as ibrutinib at 50 mg/kg QD, its clinical relevant dose. In the systemic model, the median survival of BGB-3111 25 mg/kg BID group was significantly longer than those of both ibrutinib 50 mg/kg QD and BID groups. In an ABC-subtype DLBCL (TMD-8) subcutaneous xenograft model, BGB-3111 also demonstrated better anti-tumor activity than ibrutinib. Preliminary 14-day toxicity study in rats showed that BGB-3111 was very well tolerated and maximal tolerate dose (MTD) was not reached when it was dosed up to 250mg/kg/day. In conclusion, BGB-3111 is a highly selective and potent BTK inhibitor. It does not affect rituximab-induced ADCC and demonstrated better efficacy than ibrutinib in xenograft models, supporting further clinical investigation of this compound both as single agent and in combination with anti-CD20 antibodies in hematological cancers. Citation Format: Na Li, Zhijian Sun, Ye Liu, Mingming Guo, Yilu Zhang, Dongping Zhou, Bo Zhang, Dan Su, Shuo Zhang, Jing Han, Yajuan Gao, Yunhang Guo, Zhiwei Wang, Min Wei, Lusong Luo, Lai Wang. BGB-3111 is a novel and highly selective Bruton's tyrosine kinase (BTK) inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2597. doi:10.1158/1538-7445.AM2015-2597

The PI3K/AKT/mTOR signaling pathway plays a fundamental role in cell proliferation, growth and survival and aberrant activation of this signaling pathway has been shown to drive the progression of malignant tumors.[1] Drugs targeting the pathway at multiple points, such as dual PI3K/mTOR inhibitors appear to have the broadest activity profile to address cancer therapeutic strategies and are currently being explored in numerous clinical studies. Recently, we presented PQR309, a novel, brain-penetrant pan-PI3K/mTOR inhibitor, which entered phase II clinical trials in 2016.[2] Here, we report the lead optimization of PQR530, a potent and brain-penetrant follow-up compound as pan-PI3K/mTORC1/2 inhibitor. The development of a follow-up compound concentrated on the improvement of both, the potency and the selectivity for all targeted kinases, namely the class IA PI3K isoforms as well as mTOR. We present a detailed ligand-based structure-activity relationship study which was obtained by systematic modifications of the hinge region as well as the affinity binding substituents. This study led to the identification of PQR530, a dual pan-PI3K/mTORC1/2 inhibitor showing excellent activities in cellular assays as well as in PI3Kα and mTOR enzymatic binding assays. In A2058 melanoma cells PQR530 inhibited protein kinase B (PKB, pSer473) and ribosomal protein S6 (pS6, pSer235/236) phosphorylation with IC50 values of 0.07 µM. PQR530 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR530 displayed potency in a panel of 44 cancer cell lines (NTRC OncolinesTM) to prevent cancer cell growth (mean value for GI50 of 426 nM). Oral application of PQR530 to mice resulted in a dose-proportional PK and demonstrated good oral bioavailability and excellent brain penetration.[3] An optimized, robust synthetic route allowed rapid access to multi-gram quantities of PQR530 for pre-clinical development in only 4 steps. In conclusion, PQR530 inhibits all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 potently and selectively, and shows anti-tumor effects in vitro and in vivo. [1] M. P. Wymann, M. Zvelebil, M. Laffargue (2003). Phosphoinositide 3-kinase signalling – which way to target? Trends Pharmacol Sci.; 24, 366-376. [2] V. Cmiljanovic et. al. “PQR309: Structure-Based Design, Synthesis and Biological Evaluation of a Novel, Selective, Dual Pan-PI3K/mTOR Inhibitor” presented at AACR Annual Meeting 2015, April 18-22, Philadelphia, Pennsylvania, USA. [3] P. Hillmann et al. “Pharmacological Characterization of the Selective, Orally Bioavailable, Potent Dual PI3K/mTORC1/2 Inhibitor PQR530” abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Denise Rageot, Florent Beaufils, Anna Melone, Alexander M. Sele, Thomas Bohnacker, Marc Lang, Jürgen Mestan, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2017-140

Diacylglycerol kinases (DGKs), a family of isozymes that phosphorylate the membrane lipid, diacylglycerol (DAG), to phosphatidic acid (PA), are important players in signal transduction cascades. DAG and PA act as vital second messengers that regulate multiple cellular signal transduction pathways including PKC and MAPK. DGKα (DGKA), one of the ten human DGK isoforms, has been reported to play a role in mediating numerous aspects of cancer progression including survival, migration, and invasion of cancer cells. Emerging data indicate that DGKA mediates T-cell dysfunction during anti-PD-1 therapy, playing a role in the development of resistance to PD-1 blockade. This suggests that DGKA inhibition offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer. Although serotonin (5-HT) antagonists ritanserin, R59022 and R59949 have been previously repurposed as DGKA inhibitors, their potency and selectivity profiles are largely unsatisfactory. Therefore, there is a significant unmet clinical need to develop potent and selective DGKA inhibitors. Here, we used our proprietary PandaOmics platform to explore targets that can overcome the resistance to anti-PD1 therapy. In order to do so, we created a project that included bulk RNAseq data from 4 datasets alongside with 2 single-cell RNAseq datasets. Based on the calculation results, DGKA inhibitors may overcome anti-PD-1 resistance and expand the responder patient population in cancer immunotherapy. We then designed and evaluated a series of compounds in biochemical and cellular assays, and report that ISM4312A is a novel DGKA inhibitor with excellent potency (IC50 of 120 pM) and high selectivity (>25000 fold) for DGKA versus 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2B. Further, ISM4312A showed high selectivity against 468 other kinases. In the cellular T-cell activation assay, ISM4312A induced IL-2 production in a dose-dependent manner and enhanced T-cell activity at concentrations as low as 20 nM. In the MC38 in vivo syngeneic model, ISM4312A at 3 mg/kg BID showed robust anti-tumor activities with (Tumor Growth Inhibition, TGI, 93.4%, 37.5% mice achieved CR) or without (TGI, 25.2%) anti-PD-1 therapy. Data from this combination study indicate synergistic activity as compared with the single anti-PD-1 treatment (TGI, 50.8% at 5 mg/kg BID, no mice achieved CR). ISM4312A also exhibited favorable ADME, excellent oral bioavailability, and tolerance. Together, the data support the ongoing clinical development of ISM4312A as a potential first-in-class DGKA inhibitor in cancer immunotherapy. Citation Format: Deheng Sun, Hongfu Lu, Huaxing Yu, Feng Wang, Mike Korzinkin, Xin Cai, Xiao Ding, Sujata Rao, Feng Ren, Alex Zhavoronkov. Targeting DGKA for immuno-oncology therapy: ISM4312A, a novel DGKA inhibitor with robust anti-tumor activity [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 1855.

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.