Hematological Cancer Cell Lines Research Articles

Ligufalimab, a novel anti-CD47 antibody with no hemagglutination demonstrates both monotherapy and combo antitumor activity

BackgroundCD47 is a widely expressed transmembrane glycoprotein that delivers an antiphagocytic signal on macrophages through its interaction with SIRPα. CD47 is highly expressed in cancer cells and its overexpression is...

HDAC inhibitors suppress protein poly(ADP-ribosyl)ation and DNA repair protein levels and phosphorylation status in hematologic cancer cells: implications for their use in combination with PARP inhibitors and chemotherapeutic drugs.

The therapeutic efficacy of histone deacetylase inhibitors (HDACi) for hematologic malignancies and solid tumors is attributed to their ability to remodel chromatin, normalize dysregulated gene expression, and inhibit repair of damaged DNA. Studies on the interactions of HDACi with PARP inhibitors in hematologic cancers are limited, especially when combined with chemotherapeutic agents. Exposure of hematologic cancer cell lines and patient-derived cell samples to various HDACi resulted in a significant caspase-independent inhibition of protein PARylation, mainly catalyzed by PARP1. HDACi affected the expression of PARP1 at the transcription and/or post-translation levels in a cell line-dependent manner. HDACi-mediated inhibition of PARylation correlated with decreased levels and phosphorylation of major proteins involved in DNA repair. Combination of HDAC and PARP1 inhibitors provided synergistic cytotoxicity, which was further enhanced when combined with a chemotherapeutic regimen containing gemcitabine, busulfan and melphalan as observed in lymphoma cell lines. Our results indicate that the anti-tumor efficacy of HDACi is partly due to down-regulation of PARylation, which negatively affects the status of DNA repair proteins. This repair inhibition, combined with the high levels of oxidative and DNA replication stress characteristic of cancer cells, could have conferred these hematologic cancer cells not only with a high sensitivity to HDACi but also with a heightened dependence on PARP and therefore with extreme sensitivity to combined HDACi/PARPi treatment and, by extension, to their combination with conventional DNA-damaging chemotherapeutic agents. The observed synergism of these drugs could have a major significance in improving treatment of these cancers.

292 (PB072) - Development of selective MCL-1 heterobifunctional degraders

Background: MCL1 is a member of the Bcl-2 family that plays a key role in cellular homeostasis through the regulation of apoptosis as well as other less characterized functions. The growing recognition of MCL-1 role in cancer cell survival and its association with the development of anticancer drug resistance makes it an attractive target for cancer therapy. Several inhibitors have been developed during the past decade and some of them have entered clinical trials, but no drugs have been approved for clinical use so far. Targeted protein degradation using heterobifunctional degraders has emerged as a novel therapeutic modality in drug discovery. This technology has many potential advantages over traditional inhibitors, e.g. reduced side effects and drug resistance, extended duration of action, elevated selectivity, and the opportunity to target “undruggable” proteins. In this study, we report the development of the first-in-class MCL-1 bifunctional degraders with in vivo PD effect. Material and Methods: Compounds’ affinity to CRBN (Cereblon), an E3 ligase, and to MCL-1 was determined by FP and SPR, respectively. AlphaLISA and HTRF were utilized to confirm ternary complex formation. Cell viability was assessed by CellTiter-Glo Assay. Apoptosis induction and changes in mitochondrial membrane potential were assessed by flow cytometry using Annexin/PI and TMRE staining, respectively. Targeted protein degradation in cells treated with compounds in the absence or presence of apoptosis inhibitor was assessed by WB. The mechanism of action of the compounds was confirmed in cancer cells pre-treated with proteasome or NEDD8-Activating Enzyme inhibitors, or CRBN-targeting siRNA, and then treated with the compounds. MCL-1 abundance and expression of apoptosis markers were confirmed by WB. PK studies were conducted in CD1 mice and Sprague Dawley rats, and PD studies in CB-17 SCID mice xenografted with MV4–11 cells. Results: Synthesized compounds bind CRBN and MCL-1 with high affinity, form a ternary complex in vitro and induce the degradation of MCL-1 in cells, which is caspase- and proteasome-dependent and leads to apoptosis induction. These series of compounds showed a nM range cytotoxic activity in haematological cancer cell lines with the best compounds having an IC50 <100 nM. Moreover, MCL-1 degradation in vivo can be achieved by IP administration leading to the initiation of the programmed cell death. Conclusions: Our compounds have a broad cytotoxic activity toward cultured human cancer cells and a promising in vivo pharmacokinetic profile. Our data indicate that targeting MCL-1 by employing bifunctional degraders represents a potentially new and effective strategy for cancer treatment. No conflict of interest.

MCL-319 LP-284 - A Highly Potent Small Molecule Targeting Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL). Approximately 40%-50% of MCL patients carry inactivating mutations in the gene ataxia telangiectasia mutated (ATM), a core component of the cell's DNA damage response system. MCL cells with mutations in ATM are known to have increased chromosomal imbalance, copy number loss, and hypersensitivity to DNA-damaging agents. LP-184, a small-molecule DNA-damaging agent, is known to be synthetically lethal in tumors with DNA-repair deficiencies, including tumors with ATM mutations. However, DNA damage by LP-184 is dependent on activation by the oxidoreductase prostaglandin reductase 1 (PTGR1), which is expressed at low levels in many hematological cancers. We synthesized LP-284, an enantiomer of LP-184, which retains synthetic lethality in cancers with impaired DNA damage repair pathway genes (e.g., ERCC1, ERCC2, ERCC3, ERCC6), but LP-284's activity is independent of PTGR1 expression. We demonstrated that LP-284 has enhanced tumoricidal activity in multiple hematological cancer cell lines using a B-cell NHL panel. The IC50 values for all 6 MCL cell lines treated with LP-284 were among the lowest for all hematological cell lines tested with LP-284. These MCL cell lines included cell lines resistant to ibrutinib, zanubrutinib, venetoclax, and bortezomib. Additionally, among the 6 MCL cell lines tested, the 3 cell lines with mutations in the nucleotide excision repair (NER) or homologous recombination (HR) DNA damage repair pathways had lower LP-284 IC50 values than the 3 cell lines without NER or HR mutations. Because ATM orchestrates the activities of the NER and HR pathways, MCL patients with ATM deficiencies are likely to have better responses to LP-284 treatment. Collectively, these data indicate that LP-284 is a promising preclinical DNA-damaging agent that possesses nanomolar-range anti-tumor activities in multiple and diverse MCL cell lines, with the potential to treat MCL patients who are resistant to other therapies.

Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy.

Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Hyperthermia-stimulated tonsil-mesenchymal stromal cells suppress hematological cancer cells through downregulation of IL-6.

There are contradictory reports on the use of mesenchymal stromal cells (MSCs) in cancer therapy. Variable outcomes have been associated with several factors including cancer pathology, experimental procedure, MSC source tissue, and individual genetic differences. It is also known that MSCs exert their therapeutic effects with various paracrine factors released from these cells. The profiles of the factors released from MSCs are altered by heat shock, hypoxia, oxidative stress, starvation or various agents such as inflammatory cytokines, and their therapeutic potential is affected. In this study, the antitumor potential of conditioned media (CM), which contains paracrine factors, of mild hyperthermia-stimulated mesenchymal stromal cells derived from lymphoid organ tonsil tissue (T-MSC) was investigated in comparison with CM obtained from T-MSCs grew under normal culture conditions. CM was obtained from T-MSCs that were successfully isolated from palatine tonsil tissue and characterized. The cytotoxic effect of CM on the growth of hematological cancer cell lines at different concentrations (1:1 and 1:2) was demonstrated by methylthiazoldiphenyl-tetrazolium bromide analysis. In addition, the apoptotic effect of T-MSC-CM treatment was evaluated on the cancer cells using Annexin-V/PI detection method by flow cytometry. The pro/anti-apoptotic and cytokine-related gene expressions were also analyzed by real-time polymerase chain reactionpost T-MSC-CM treatment. In conclusion, we demonstrated that the factors released from hyperthermia-stimulated T-MSCs induced apoptosis in hematological cancer cell lines in a dose-dependent manner. Importantly, our results at the transcriptional level support that the factors and cytokines released from hyperthermia-stimulated T-MSC may exert antitumoral effects in cancer cells by downregulation of IL-6 that promotestumorigenesis. These findings reveal that T-MSC-CM can be a powerful cell-free therapeutical strategy for cancer therapy.

3-Chloro-3-methyl-2,6-diarylpiperidin-4-ones as Anti-Cancer Agents: Synthesis, Biological Evaluation, Molecular Docking, and In Silico ADMET Prediction.

Piperidine pharmacophore-containing compounds have demonstrated therapeutic efficacy against a range of diseases and are now being investigated in cancer. A series of 3-chloro-3-methyl-2,6-diarylpiperidin-4-ones, compounds (I–V) were designed and synthesized for their evaluation as a potential anti-cancer agent. Compounds II and IV reduced the growth of numerous hematological cancer cell lines while simultaneously increasing the mRNA expression of apoptosis-promoting genes, p53 and Bax. Molecular docking analyses confirmed that compounds can bind to 6FS1, 6FSO (myeloma), 6TJU (leukemia), 5N21, and 1OLL (NKTL). Computational ADMET research confirmed the essential physicochemical, pharmacokinetic, and drug-like characteristics of compounds (I–V). The results revealed that these compounds interact efficiently with active site residues and that compounds (II) and (V) can be further evaluated as potential therapeutic candidates.

Abstract 5662: Targeting N-myristoylation for therapy of adult acute myeloid leukemia

Abstract Two N-myristoyltransferases (NMTs) NMT1 and NMT2 catalyze the reaction. NMT1 is ubiquitous expressed and is essential for cell survival while NMT2 is more variably expressed and non-essential suggesting that their substrate specificity and activity levels differ. Historically, inhibition of myristoylation was suggested as a therapeutic anti-cancer target since NMTs expression were shown to be increased in numerous types of cancers and myristoylation was shown to be essential for proper localization and activity of some important proto-oncogenes such as Src Family Kinases (SFKs). Recently, we showed NMT2 expression is lost in numerous haematological cancer cell lines (including AML) and that these haematological cancer cell lines are exquisitely sensitive to the pan-NMT inhibitor PCLX-001. PCLX-001 recently entered human clinical trials as once daily oral therapy for relapsed/refractory B-cell Non-Hodgkin Lymphoma and advanced solid malignances. Dysregulation and oncogenic activity of SFKs occurs frequently in AML, suggesting NMT inhibition could provide therapeutic benefit in this indication. Data analysis from the TCGA transcriptome database revealed that high NMT1 and low NMT2 were associated with reduced overall and event-free survival in adult AML. Moreover, high NMT1 - but not NMT2 - expression is associated with proliferative gene sets in AML cell lines. AML cell lines treated with PCLX-001 showed a significant reduction in total protein myristoylation, reduced levels of SFK proteins and SFK phosphorylation as well as significant increases in ER stress marker BIP protein and caspase 3 cleavage. PCLX-001 induced apoptosis in AML cell lines and patient blasts at concentrations that spared a large proportion of peripheral blood lymphocytes and monocytes from healthy individuals. PCLX-001 monotherapy had dose-dependent anticancer activity in an AML MV-4-11 cell line derived xenograft (CDX) and two AML patient derived xenografts (PDXs) and produced complete remissions in subcutaneous AML CDX. In tail-vein injected PDX models, PCLX-001 treatment resulted in up to 95% reduction of human CD45+ cells in peripheral blood and bone marrow. PCLX-001 preferentially targeted AML cells inducing apoptosis and reducing leukemic burden. These findings validate NMT inhibition as a novel therapeutic strategy for AML and warrant the evaluation of PCLX-001 in clinical trials for adult AML. Citation Format: Jay Gamma, Aishwarya Iyer, Megan Yap, Zoulika Zak, Krista Vincent, Cassidy Ekstrom, Qiang Liu, Erwan Beauchamp, Lynne Postovit, Jean Wang, John R. Mackey, Naveen Pemmaraju, Gautam Borthakur, Joseph Brandwein, Luc Berthiaume. Targeting N-myristoylation for therapy of adult acute myeloid leukemia [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 5662.

Abstract 3276: Overcoming immune checkpoint blockade resistance via EZH1/2 dual inhibition by HM97662 in KRAS/LKB1 mutated NSCLC

Abstract Immune checkpoint blockades (ICBs) are known as a promising treatment option against in advanced non-small-cell lung cancer (NSCLC). And KRAS is the most frequently mutated oncogene in NSCLC. However, KRAS mutation is not a significantly associated with survival benefit of ICBs. Mutations in LKB1 (aka STK11) and frequently co-occurring KRAS mutations are accompanied with poor survival in metastatic NSCLC immuno-oncology trials. Even tumor mutational burden is a proposed-potential biomarker for response to ICBs, there are therapeutic unmet needs in NSCLC patient with KRAS/LKB1 (KL) mutation. Epigenetic regulation lead to enhanced anti-tumor immunity of innate immune responses via inducing type I interferons. Also it drives to enhance adaptive immune responses by targeting T cell recognition factors as well as recruitment immune cells to tumor microenvironments (TMEs). Thus, we demonstrated the pharmacological inhibition of Enhancer of zeste homolog (EZH)1/2 enhance the efficacy of clinically available immunotherapies in KRAS/LKB1 mutated NSCLC. Previously, we reported that a novel EZH1/2 dual inhibitor, HM97662, has showed potently decreased global H3K27me3 and strong anti-proliferative activities against various hematological cancer cell lines with EZH2 activating mutations as well as solid tumor cell lines with negatively mutated components of regulatory protein complexes. In this study, we evaluated that HM97662 could induce of STING expression and STING-dependent cytokine production in KL mutant NSCLC cell lines. Furthermore, we explored anticancer effect by inducing immune modulation by EZH2 inhibitors in combination with an anti-PD-(L)1 agent was evaluated. As a result, HM97662 potently restored STING expression in KL mutated cell lines such as A549 and H460, but did not KRAS/p53 (KP) mutated H358 cells. HM97662 also dose-dependently increased the mRNA expression of CXCL10, IFN-α and IFN-β and secretion of CXCL10, IFN-α, CCL2, CCL3 and CCL5 at nanomolar concentrations in H460 cells. In accordance with this, HM97662 showed potent growth inhibition against H460 cells under co-culture condition with activated T cells. Moreover, HM97662 had superior anti-proliferation efficacy among the tested compounds in H460 cells with activated T cells, when it combined with anti-PD-L1 antibody avelumab. In our exploratory study, HM97662 increments the sensitivity of immune checkpoint inhibitor by restoration of STING expression and STING pathway related chemo/cytokine production in KRAS/LKB1 mutated NSCLC cell lines. These results demonstrate that HM97662 drives anti-cancer effect by immune modulation in combination with anti-PD-(L)1 agent, suggesting therapeutic potency to combination with ICBs by turning non-inflamed to inflamed TME. Clinical trials to prove the effectiveness of HM97662 identified in preclinical studies need to be carried out immediately. Citation Format: Jooyun Byun, DongJin Hong, Miyoung Lee, Soonho Kweon, Seung Hyun Jung, Yu-Yon Kim, Hyunjin Park, Junghwa Park, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. Overcoming immune checkpoint blockade resistance via EZH1/2 dual inhibition by HM97662 in KRAS/LKB1 mutated NSCLC [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 3276.

Abstract ND14: Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers

Abstract Pharmacological profile and anti-tumor properties of FHD-286, a novel BAF inhibitor for the treatment of transcription factor-driven cancersMartin Hentemann, Rishi G. Vaswani, Lan Xu, Richard C. Centore, Luis M. M. Soares, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Sarah Reilly, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Liyue Huang, Jessica Piel, Johannes Voigt, David S. Millan, Ho Man Chan, Ryan G. Kruger, Carl P. Decicco, Samuel Agresta, Steven F. BellonFoghorn Therapeutics, 500 Technology Square, Suite 700, Cambridge, MA 02139The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a compound that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that this SMARCA4/SMARCA2 inhibitor acts through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. FHD-286 is orally bioavailable and demonstrates robust efficacy in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. In preclinical efficacy and toxicology studies, this first in class molecule demonstrates an acceptable therapeutic index which should allow for flexibility in clinical designs. FHD-286 is currently in two Phase I trials for both relapsed refractory AML and MDS, and metastatic uveal melanoma. Citation Format: Murphy Hentemann. Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers [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 ND14.

Abstract 1397: A phenotypic-based drug discovery approach against tumors with MTAP loss

Abstract The Methylthioadenosine phosphorylase (MTAP) gene is in chromosome 9q21 location, which includes p16/CDKNA and is homozygously deleted in about 15% of all cancers, including a large proportion of gliomas, mesotheliomas and T cell lymphomas. MTAP cleaves methylthioadenosine (MTA), a byproduct of polyamine synthesis, to regenerate methionine and adenine. Several functional genomics screens have identified the downstream genes MAT2A and PRMT5 as synthetically lethal with MTAP loss. These two targets have been the subject of several drug discovery efforts. Our approach was based on a forward chemical genetic approach, in which we employed two isogenic cell lines, PANC1, with MTAP loss and PAM cells where MTAP is re-introduced. A library of over 200,000 compounds was screened for hits that affected the viability of PANC1 cell, whereas spared PAM cells. One hit series was found to reproducibly inhibit the proliferation of PANC1 cells with EC50s in the low single digit uM range and one order of selectivity over PAM cells. Phenotype-based optimization resulted in improved analogs, with prototype MTB-22252 exhibiting an EC50 of 47nM in PANC1 cells and 15-fold selectivity. An affinity probe was designed based on an analog of MTB-22252 bearing a biotin moiety via a linker and an azide group for photoactivatable cross-linking with potential interacting proteins. This probe maintained cellular activity and selectivity and it was used in an MS-proteomics experiment in PANC1 cells. The top protein identified by this experiment showed high enrichment over DMSO control and was effectively competed out by MTB-22252. MTB2252 and analogs thermo-stabilized this protein by up to 50C in PANC1 cells. PANC1 cells expressed high levels of this protein, whereas insensitive PAM cell did not express it at all. In additional cell line pairs, sensitivity to MTB-2252 correlated well with the expression levels of this protein. Reintroduction of MTAP in cells lacking MTAP and this new protein, resulted in loss of expression of the new protein, suggesting a regulatory connection. In a wider cell panel of over 130 cell lines, there was correlation of sensitivity with MTAP loss and even better correlation with the expression levels of this protein. Some of the most sensitive cell lines included several colorectal, glioma and hematologic cancer cell lines. In vivo evaluation of MTB-9655 against some of the most sensitive tumor cell lines is on-going. Citation Format: Andreas Goutopoulos, Iris Amanati, Avital Hay-Koren, Ali Fattaey, Philippe Nakache, Dimitri Kovalerchik, Dikla Paz, Simone Botti, Hanna Oppenheimer, Omri Erez, Irit Fainer. A phenotypic-based drug discovery approach against tumors with MTAP loss [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 1397.

Abstract 3696: GS-9716: A potent, selective and orally bioavailable small molecule inhibitor of MCL1 for the treatment of cancer

Abstract MCL1 is an anti-apoptotic member of the BCL2 family of proteins. These anti-apoptotic proteins prevent caspase-mediated cell apoptosis by binding to the pro-apoptotic proteins BIM, BAK and BAX, thereby inhibiting the formation of pores on the outer mitochondrial membrane. In cancer, MCL1 is upregulated to overcome such stress-induced effects, promoting cell survival, therapy resistance, and tumor progression. Here, we describe the in vitro and in vivo activity of GS-9716, a potent and selective MCL1 small molecule inhibitor that binds directly to MCL1 and induces rapid apoptosis in cancer cells by activating the mitochondrial apoptotic pathway. GS-9716 displayed potent and selective disruption of human MCL1-BIM protein dimer in vitro. In hematological and solid tumor cell line models, GS-9716 led to dose dependent reduction of MCL1-BIM and MCL1-BAK protein dimers. MCL1 protein dimer (BAK and BIM) reduction correlated with dose-dependent increases in cleaved caspase (apoptosis marker). GS-9716 potently reduced cellular viability across panels of hematological (Median GI50 = 30 nM) and breast cancer cell lines (Median GI50 = 470 nM). GS-9716 exhibits favorable pharmacokinetics properties with good oral bioavailability across species. GS-9716 dosed daily, once per week, or 7-day cycles of 2 days on/5 days off, resulted in significant tumor growth inhibition against models of multiple myeloma (H929), several TNBC cell line xenografts (HCC1187 and HCC70) and patient-derived xenografts. Also, in these models, GS-9716 demonstrated dose-dependent MCL1 protein dimer (BIM and BAK) reduction and activation of apoptosis in tumors. In addition to single agent activity, GS-9716 showed strong synergy with multiple anti-cancer therapies; robust in vitro synergy was observed with venetoclax in the AML models and as well as with paclitaxel in the TNBC models. The combination treatment of paclitaxel (dosed IV, QW) with GS-9716 (dosed orally, 2 days on/5 days off) in the TNBC HCC1187 xenograft model achieved complete tumor regression. Similar responses were also observed when GS-9716 was dosed with paclitaxel in the TNBC PDX models. GS-9716 is currently in Phase 1 clinical trial evaluating safety, tolerability and pharmacokinetics as monotherapy and in combination with anticancer therapies in adults with advanced solid tumors (NCT05006794). Citation Format: Clinton K. Matson, Thomas Kenney, Bart H. Steiner, Chloe R. Deodato, Anella Yahiaoui, Claudia A. Rubio, Julie T. Chan, Nevena Mollova, Kathleen S. Keegan, Chandrasekar Venkataramani. GS-9716: A potent, selective and orally bioavailable small molecule inhibitor of MCL1 for the treatment of 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 3696.

A TCR mimic CAR T cell specific for NDC80 is broadly reactive with solid tumors and hematologic malignancies

AbstractTarget identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02–restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

Abstract P020: Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor

Abstract Background: Ribonucleotide reductase (RNR) plays a crucial role in dNTP biosynthesis, which is required for DNA synthesis and repair, and is thought to be an attractive cancer therapeutic target. However, the precise significance of RNR inhibition remains to be elucidated, since reported RNR inhibitors exhibit limited pharmacological potency and off-target effects. We have developed a highly potent and novel small molecule RNR inhibitor, TAS1553, and reported that TAS1553 disrupted the protein-protein interaction between RNR subunits and exhibited the broad antiproliferative activity against human cancer cells in both in vitro and in vivo via oral administration. Phase-I study is currently ongoing and the identification of a predictive biomarker is essential to maximize clinical benefit of TAS1553. Here, we report a predictive biomarker of the response to TAS1553, and a clinical development strategy with the biomarker use. Material and methods: TAS1553 was synthesized at Taiho Pharmaceutical Co., Ltd. DNA replication stress, apoptosis, caspase activity and cellular growth inhibition induced by TAS1553 were assessed by western blotting, immunofluorescence staining, Caspase-Glo 3/7 assay and CellTiter-Glo® 2.0 assay, respectively. Cells were transfected with 2 nM siRNAs against SLFN11 (siSLFN11) and used for caspase-3/7 activation assay and cell proliferation assay. Results: TAS1553 induced intracellular pChk1, pRPA2 and γH2AX, followed by cleavage of PARP and caspase-3, suggesting that TAS1553 causes massive DNA replication stress. Then, we explored factors, involved in DNA replication stress, to predict response to TAS1553. Cytotoxicity profiling revealed broad antiproliferative activity of TAS1553 against both human hematological and solid cancer cell lines in a dose-​dependent manner (GI50 = 228-4150 nmol/L), and global gene expression profiling revealed that cells with high SLFN11 mRNA expression showed a higher sensitivity to TAS1553. TAS1553 exerted growth inhibitory activity without any cell killing effect against cells with low SLFN11 expression even at a concentration of 10 μmol/L, but cell killing activity against cells with high SLFN11 expression. Depletion of SLFN11 by siSLFN11 treatment in A673 cells which have high SLFN11 expression suppressed cytotoxic effect but not the growth inhibitory effect of TAS1553. Furthermore, we observed suppression of caspase-3/7 activation induced by TAS1553 in A673 cells transfected with siSLFN11. SLFN11 appeared to sensitize cancer cells to TAS1553 via promoting apoptosis. Conclusions: TAS1553, a novel orally available RNR inhibitor, showed potent antitumor activity in preclinical models of both hematological and solid tumors. Thus, TAS1553 could be a promising therapeutic agent for cancer, and SLFN11 could be a predictive biomarker in order to maximize clinical response to TAS1553. Citation Format: Hiroto Fukushima, Hiroyuki Ueno, Takuya Hoshino, Wakako Yano, Hiraku Itadani, Miki Terasaka, Sayaka Tsukioka, Takamasa Suzuki, Shoki Hara, Yoshio Ogino, Khoon Tee Chong, Tatsuya Suzuki, Yoshihiro Otsu, Satoshi Ito, Nozomu Tanaka, Seiji Miyahara. Schlafen 11 (SLFN11) as a predictive biomarker of the response to TAS1553, a novel small molecule ribonucleotide reductase subunit interaction inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P020.

Abstract P237: PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification

Abstract Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.

The Antileukemic Effect of Xestoquinone, A Marine-Derived Polycyclic Quinone-Type Metabolite, Is Mediated through ROS-Induced Inhibition of HSP-90.

Xestoquinone is a polycyclic quinone-type metabolite with a reported antitumor effect. We tested the cytotoxic activity of xestoquinone on a series of hematological cancer cell lines. The antileukemic effect of xestoquinone was evaluated in vitro and in vivo. This marine metabolite suppressed the proliferation of Molt-4, K562, and Sup-T1 cells with IC50 values of 2.95 ± 0.21, 6.22 ± 0.21, and 8.58 ± 0.60 µM, respectively, as demonstrated by MTT assay. In the cell-free system, it inhibited the activity of topoisomerase I (Topo I) and II (Topo II) by 50% after treatment with 0.235 and 0.094 μM, respectively. The flow cytometric analysis indicated that the cytotoxic effect of xestoquinone was mediated through the induction of multiple apoptotic pathways in Molt-4 cells. The pretreatment of Molt-4 cells with N-acetyl cysteine (NAC) diminished the disruption of the mitochondrial membrane potential (MMP) and apoptosis, as well as retaining the expression of both Topo I and II. In the nude mice xenograft model, the administration of xestoquinone (1 μg/g) significantly attenuated tumor growth by 31.2% compared with the solvent control. Molecular docking, Western blotting, and thermal shift assay verified the catalytic inhibitory activity of xestoquinone by high binding affinity to HSP-90 and Topo I/II. Our findings indicated that xestoquinone targeted leukemia cancer cells through multiple pathways, suggesting its potential application as an antileukemic drug lead.

The Positive Enantiomer of a Novel Chiral DNA Alkylating Agent Exhibits Nanomolar Potency in Hematologic Cancers

LP-184, or (-)-hydroxyurea methylacylfulvene, is a potent DNA alkylating agent that effectively kills solid tumors. It belongs to the acylfulvene compound family known to induce DNA lesions repaired by the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. Here, we show that LP-284, the synthetic positive enantiomer of LP-184, exhibited the greatest and broadest hematologic cancer antiproliferative activities among the 6 acylfulvenes, including illudin S, illudin M, Irofulven (LP-100), the semisynthetic racemic LP-184, the synthetic negative enantiomer LP-184, and LP-284. The distinct pharmacological activities of LP-284 may be due to differences in metabolic activation, transport, or affinity to cellular macromolecules. To determine whether metabolic activation plays a role, we compared the correlation between the expression of Prostaglandin Reductase 1 (PTGR1), the NADPH-dependent oxidoreductase known to convert Irofulven into its active metabolite, and the IC50 of LP-184, Irofulven, and LP-284. We found that the expression level of PTGR1 is highly correlated with LP-184 (r=0.88, p=8.4e-20) and Irofulven (r=0.71, p=4.7e-10) sensitivity, but not with LP-284 (r=-0.01, p=0.93). We also found that the average expression level of PTGR1 is significantly lower in hematologic cancer cell lines (n=180) than in solid tumor cell lines (n=856), indicating the existence of an alternative LP-284 activator in hematologic cells. Next, we checked mutation status, RNA expression, protein expression, and DNA methylation of 489 oxidoreductases, but none of the enzymes was highly correlated with LP-284 activity.To further explore the potential clinical application of LP-284 in hematologic cancers, we conducted cell viability assays in 18 hematologic cancer cell lines and found that LP-284 exhibited nanomolar potency in acute lymphocytic leukemia (average IC50: 351 nM), chronic myeloid leukemia (average IC50: 360 nM), B-cell lymphoma (average IC50: 366 nM), and Multiple Myeloma (MM, IC50: 334 nM). We also investigated the therapeutic potential of LP-284 in combination with spironolactone in treating MM. Spironolactone, an FDA approved drug for hypertension, degrades one of the key TC-NER players ERCC3 in MM, which in turn makes cells more vulnerable to helix-distorting DNA lesions likely caused by LP-284. While Spironolactone alone didn't cause cytotoxicity to the MM cell line RPMI8226, it reduced LP-284 IC50 by 2.4 fold.Taken together, we have demonstrated the importance of stereochemistry in acylfulvene activity. LP-284, likely to be activated through a different route, is a unique and potent acylfulvene for hematologic cancers. Additionally, pharmacological inhibition of the TC-NER pathway greatly promoted LP-284 cytotoxicity. We hypothesize that LP-284 induces DNA lesions, which may be lethal to TC-NER deficient cells and may block transcription of short-lived fusion genes that are essential for cancer cell survival until repaired. Therefore, our discovery of the novel enantiomer LP-284 may provide a targeted therapy option for hematologic cancers with compromised DNA repair. DisclosuresZhou: Lantern Pharma: Current Employment. Biyani: Lantern Pharma: Current Employment. Kathad: Lantern Pharma: Current Employment, Current equity holder in publicly-traded company. Kulkarni: Lantern Pharma: Current Employment. McDermott: Lantern Pharma: Current Employment. Bhatia: Lantern Pharma: Current Employment.

Antitumor activity of novel POLA1-HDAC11 dual inhibitors

Hybrid molecules targeting simultaneously DNA polymerase α (POLA1) and histone deacetylases (HDACs) were designed and synthesized to exploit a potential synergy of action. Among a library of screened molecules, MIR002 and GEM144 showed antiproliferative activity at nanomolar concentrations on a panel of human solid and haematological cancer cell lines. In vitro functional assays confirmed that these molecules inhibited POLA1 primer extension activity, as well as HDAC11. Molecular docking studies also supported these findings. Mechanistically, MIR002 and GEM144 induced acetylation of p53, activation of p21, G1/S cell cycle arrest, and apoptosis. Oral administration of these inhibitors confirmed their antitumor activity in in vivo models. In human non-small cancer cell (H460) xenografted in nude mice MIR002 at 50 mg/kg, Bid (qd × 5 × 3w) inhibited tumor growth (TGI = 61%). More interestingly, in POLA1 inhibitor resistant cells (H460-R9A), the in vivo combination of MIR002 with cisplatin showed an additive antitumor effect with complete disappearance of tumor masses in two animals at the end of the treatment. Moreover, in two human orthotopic malignant pleural mesothelioma xenografts (MM473 and MM487), oral treatments with MIR002 and GEM144 confirmed their significant antitumor activity (TGI = 72–77%).Consistently with recent results that have shown an inverse correlation between POLA1 expression and type I interferon levels, MIR002 significantly upregulated interferon-α in immunocompetent mice.

Multimeric Anti-DR5 IgM Agonist Antibody IGM-8444 Is a Potent Inducer of Cancer Cell Apoptosis and Synergizes with Chemotherapy and BCL-2 Inhibitor ABT-199.

Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro, a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition.