Hematological Cancer Cell Research Articles

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 4114: Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of cancers

Abstract Background: E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed acetyl transferases (HAT) that also act as co-activators for number of transcription factors including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Both CBP and p300 possess bromodomain (BRD) and a lysine acetyltransferase (KAT) domain. These two closely related epigenetic modulators are known to play an oncogenic role in a variety of cancers. Functional synthetic lethal screens have identified preferential killing in CBP-deficient and/or MYC-dependent hematological cancer cells by suppression of the paralogue p300. CBP/p300 BRD inhibitor could also prevent AR and ER signaling, thereby potentially inhibiting growth of AR and ER -dependent cancer cells. Thus, targeting CBP/p300 represents an attractive approach for developing personalized cancer therapies. Experimental Results: Multiple potent and selective CBP/p300 BRD inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The lead compound AU-18069 was optimized towards attaining good potency, selectivity, physicochemical properties and DMPK profile. AU-18069 potently inhibited proliferation of a wide range of cell lines derived from prostate cancer, breast cancer and CBP mutant/MYC-dependent hematological cancers. The lead compound demonstrated good PK profile in rodents as well as in dogs. Excellent anti-tumor efficacy was achieved in leukemia and lymphoma xenograft models at well-tolerated doses. Significant downregulation of c-Myc was observed in a single dose PK-PD study. Conclusion: Lead candidate AU-18069 demonstrated that selective CBP/p300 bromodomain inhibitors are efficacious in models of hematologic malignancies and solid cancers in vitro and in vivo. Further evaluation of efficacy in other xenograft models, as a single agent as well as in combination with other agents is planned. Long term toxicological and safety pharmacology evaluation in different species and other IND enabling studies are in progress to support progression of this compound to clinical trials. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Aravind A B, Amit A Dhudashiya, Sivapriya Marappan, Naveen Kumar R, Raghavendra N R, Mamon Dey, Avinash Kumar, Uma Bharathi B V, Girish Aggunda Renukappa, N Venkata Siva Reddy, Asha Babu, Aakanksha J Shetty, Amith A, Narasimha Rao K, Suraj T Gore, Mahaboobi Jaleel, Ramesh S Senaiar, Vijaya Shankar Nataraj, Subhendu Mukherjee, Samiulla D S, Thomas Antony, Girish Daginakatte, Rajesh Eswarappa, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Evaluation of AU-18069, a novel small molecule CBP/p300 bromodomain inhibitor for the treatment of 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 4114.

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.

Structural Feature Analyzation Strategies toward Discovery of Orally Bioavailable PROTACs of Bruton’s Tyrosine Kinase for the Treatment of Lymphoma

Bruton's tyrosine kinase proteolysis-targeting chimeras (BTK-PROTACs) have emerged as a promising approach to address the limitations of BTK inhibitors. However, conducting the rational discovery of orally bioavailable BTK-PROTACs presents significant challenges. In this study, dimensionality reduction analysis and model molecule validation were utilized to identify some key structural features for improving the oral absorption of BTK-PROTACs. The results were applied to optimize the newly discovered BTK-PROTACs B1 and B2. Compound C13 was discovered with improved oral bioavailability, high BTK degradation activity, and selectivity. It exhibited inhibitory effects against different hematologic cancer cells and attenuated the BTK-related signaling pathway. The oral administration of C13 effectively reduced BTK protein levels and suppressed tumor growth. This study led to the discovery of a new orally bioavailable BTK-PROTAC for the treatment of lymphoma, and we hope that the strategy will find wide utility.

Retention of Donor T Cells in Lymphohematopoietic Tissue and Augmentation of Tissue PD-L1 Protection for Prevention of GVHD While Preserving GVL Activity.

Allogeneic hematopoietic cell transplantation (Allo-HCT) is a curative therapy for hematological malignancies (i.e., leukemia and lymphoma) due to the graft-versus-leukemia (GVL) activity mediated by alloreactive T cells that can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells can cause a serious side effect, known as graft-versus-host disease (GVHD). GVHD and GVL occur in distinct organ and tissues, with GVHD occurring in target organs (e.g., the gut, liver, lung, skin, etc.) and GVL in lympho-hematopoietic tissues where hematological cancer cells primarily reside. Currently used immunosuppressive drugs for the treatment of GVHD inhibit donor T cell activation and expansion, resulting in a decrease in both GVHD and GVL activity that is associated with cancer relapse. To prevent GVHD, it is important to allow full activation and expansion of alloreactive T cells in the lympho-hematopoietic tissues, as well as prevent donor T cells from migrating into the GVHD target tissues, and tolerize infiltrating T cells via protective mechanisms, such as PD-L1 interacting with PD-1, in the target tissues. In this review, we will summarize major approaches that prevent donor T cell migration into GVHD target tissues and approaches that augment tolerization of the infiltrating T cells in the GVHD target tissues while preserving strong GVL activity in the lympho-hematopoietic tissues.

How Phosphofructokinase-1 Promotes PI3K and YAP/TAZ in Cancer: Therapeutic Perspectives.

Simple SummaryWe propose that PFK1 promotes a positive feedback loop with PI3K/AKT and YAP/TAZ signaling pathways in cancer cells. Therefore, targeting PFK1 (or its product F-1,6-BP) could improve the efficacy of PI3K and YAP/TAZ inhibitors currently tested in clinical trials. To this aim, we suggest the use of citrate, which is a physiologic and potent inhibitor of PFK1.PI3K/AKT is one of the most frequently altered signaling pathways in human cancers, supporting the activation of many proteins sustaining cell metabolism, proliferation, and aggressiveness. Another important pathway frequently altered in cancer cells is the one regulating the YAP/TAZ transcriptional coactivators, which promote the expression of genes sustaining aerobic glycolysis (such as WNT, MYC, HIF-1), EMT, and drug resistance. Of note, the PI3K/AKT pathway can also regulate the YAP/TAZ one. Unfortunately, although PI3K and YAP inhibitors are currently tested in highly resistant cancers (both solid and hematologic ones), several resistance mechanisms may arise. Resistance mechanisms to PI3K inhibitors may involve the stimulation of alternative pathways (such as RAS, HER, IGFR/AKT), the inactivation of PTEN (the physiologic inhibitor of PI3K), and the expression of anti-apoptotic Bcl-xL and MCL1 proteins. Therefore, it is important to improve current therapeutic strategies to overcome these limitations. Here, we want to highlight how the glycolytic enzyme PFK1 (and its product F-1,6-BP) promotes the activation of both PI3K/AKT and YAP/TAZ pathways by several direct and indirect mechanisms. In turn, PI3K/AKT and YAP/TAZ can promote PFK1 activity and F-1,6-BP production in a positive feedback loop, thus sustaining the Warburg effect and drug resistance. Thus, we propose that the inhibition of PFK1 (and of its key activator PFK2/PFKFB3) could potentiate the sensitivity to PI3K and YAP inhibitors currently tested. Awaiting the development of non-toxic inhibitors of these enzymes, we propose to test the administration of citrate at a high dosage, because citrate is a physiologic inhibitor of both PFK1 and PFK2/PFKFB3. Consistently, in various cultured cancer cells (including melanoma, sarcoma, hematologic, and epithelial cancer cells), this “citrate strategy” efficiently inhibits the IGFR1/AKT pathway, promotes PTEN activity, reduces Bcl-xL and MCL1 expression, and increases sensitivity to standard chemotherapy. It also inhibits the development of sarcoma, pancreatic, mammary HER+ and lung RAS-driven tumors in mice without apparent toxicities.

Improved anti-tumor T cell generation using rapidly differentiated dendritic cells

Abstract After allogeneic stem cell transplantation, minor histocompatibility antigens (MiHAs), Wilms-Tumor 1 (WT1) antigen, and other peptides presented by host hematologic cancer (HC) cells can lead to the in vivo expansion of donor T cells crucial for the graft-versus-leukemia effect. While the specific ex vivo production of such leukemia-specific T cells represents an interesting treatment approach, the generation of these antigen-specific donor T cells represents a major challenge. We have developed a protocol enabling the infusion of monocyte derived dendritic cell (DC)-induced ex vivo expanded T cells specific for MiHAs, WT1 and other peptides that are preferentially expressed on hematologic cells for the treatment of relapsed HC patients. While it provides interesting results, our 42-day expansion protocol is costly and requires prolonged culture periods during which patient with HC could deteriorate rapidly. To accelerate T cell production, we compared DCs generated in 3 days (DC3) vs 9 days (DC9) using standard maturation cocktail and toll-like receptor agonist. DC3 showed small differences in expression of maturation/activation markers and a unique gene signature vs DC9. Importantly, the frequency of WT1-specific CD8 T cells, primed by DC3, was slightly higher than that in DC9 primed cultures. Upon restimulation, these DC3-primed-WT1-specific T cells expressed higher amounts of IFNγ, TNFα, and CD107a degranulation marker, and were cytotoxic against WT1-expressing autologous blast cells. Together, these data show that matured DC3 have a strong capacity to prime and expand functional anti-tumor antigen-specific CD8 T cells and thus offer most interesting features for ex vivo expansion of T cells for cancer immunotherapy.

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.

Targeting metabolism: A potential strategy for hematological cancer therapy.

Most hematological cancer-related relapses and deaths are caused by metastasis; thus, the importance of this process as a target of therapy should be considered. Hematological cancer is a type of cancer in which metabolism plays an essential role in progression. Therefore, we are required to block fundamental metastatic processes and develop specific preclinical and clinical strategies against those biomarkers involved in the metabolic regulation of hematological cancer cells, which do not rely on primary tumor responses. To understand progress in this field, we provide a summary of recent developments in the understanding of metabolism in hematological cancer and a general understanding of biomarkers currently used and under investigation for clinical and preclinical applications involving drug development. The signaling pathways involved in cancer cell metabolism are highlighted and shed light on how we could identify novel biomarkers involved in cancer development and treatment. This review provides new insights into biomolecular carriers that could be targeted as anticancer biomarkers.

Surface-Functionalized Polymeric siRNA Nanoparticles for Tunable Targeting and Intracellular Delivery to Hematologic Cancer Cells.

To date, the application of RNA therapeutics to hematologic malignancies has been challenging owing to the resistance of blood cancer cells against conventional transfection methods. Herein, triple-targeting moiety-functionalized polymeric small interfering RNA (siRNA) nanoparticles were systematically developed for efficient targeted delivery of RNA therapeutics to hematologic cancer cells. Polymeric siRNAs were synthesized using rolling circle transcription and were surface-functionalized with three types of targeting moieties─a natural ligand and two additional combinations of cell-specific antibodies─for tunable targetability. As a proof of concept, the optimization of the hyaluronic acid/antibody conjugation ratio was performed for selective intracellular delivery to various non-Hodgkin's lymphoma (NHL) cell lines (Daudi, Raji, Ramos, and Toledo cells) via receptor-mediated endocytosis. The engineered nanoparticles showed almost 10-fold enhanced NHL-specific intracellular delivery and induced significant in vitro anticancer effects. This multitargeted nanoparticle platform may effectively support the intracellular delivery of polymeric siRNA sequences, and thus promote therapeutic effects in hematopoietic malignancies.

Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.

Triptolide Shows High Sensitivity and Low Toxicity Against Acute Myeloid Leukemia Cell Lines Through Inhibiting WSTF-RNAPII Complex.

Triptolide exhibits superior and broad-spectrum antitumor activity. However, the narrow safety window caused by the toxicity of triptolide limits its clinical applications. Although several characterized targets for triptolide are reported, the association between triptolide and its targets in cancer therapy is not fully understood. Here, we show that acute myeloid leukemia (AML) cell lines are sensitive to triptolide by constructing an in vitro cell and in vivo xenograft models. Meanwhile, the triptolide-induced hepatotoxicity increases with increasing dosages within the xenograft models. Additionally, the expression levels of WSTF-RPB1 are strongly associated with the sensitivity to triptolide in hematological cancer cells and can be downregulated in a dose and time-dependent manner. Finally, we show that optimizing dosing regimens can achieve the same pharmaceutical effect and reduce toxicity. In summary, this study aims to search for triptolide-sensitive cell lines as well as the underlying molecular mechanisms in order to broaden the safety window of triptolide; thus, increasing its clinical utility.

Successful Incorporation of Exosome-Capturing Antibody-siRNA Complexes into Multiple Myeloma Cells and Suppression of Targeted mRNA Transcripts.

Simple SummaryAlthough nucleic acid medicines are expected to function as new therapeutic agents, their targeted delivery into cancer cells, particularly hematologic cancer cells, via systemic administration, is limited. Based on our previous finding that tumor cell-derived exosomes are preferentially incorporated into their parental cancer cells, we previously demonstrated that anti-CD63 monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers (9mer) were transferred into solid cancer cells and inhibited exosomal miRNA functions. To challenge the delivery of siRNAs into hematologic cancer cells, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNA) with branched Arg linkers (9+9mer). Anti-CD63 mAb-conjugated complexes were incorporated into multiple myeloma (MM) cells. Moreover, these exosome-capturing mAb-conjugated siRNAs successfully decreased the mRNA transcript levels of targeted mRNAs in the MM cells. This technology could lead to a breakthrough in drug delivery systems for hematologic cancer therapy.Nucleic acid medicines have been developed as new therapeutic agents against various diseases; however, targeted delivery of these reagents into cancer cells, particularly hematologic cancer cells, via systemic administration is limited by the lack of efficient and cell-specific delivery systems. We previously demonstrated that monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers were transferred into solid cancer cells and inhibited exosomal miRNA functions. In this study, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNAs) with branched Arg linkers and investigated their effects on multiple myeloma (MM) cells. Anti-CD63 mAb-conjugated siRNAs were successfully incorporated into MM cells. The incorporation of exosomes was inhibited by endocytosis inhibitors. We also conducted a functional analysis of anti-CD63 mAb-conjugated siRNAs. Ab-conjugated luciferase+ (luc+) siRNAs significantly decreased the luminescence intensity in OPM-2-luc+ cells. Moreover, treatment with anti-CD63 mAb-conjugated with MYC and CTNNB1 siRNAs decreased the mRNA transcript levels of MYC and CTNNB1 to 52.5% and 55.3%, respectively, in OPM-2 cells. In conclusion, exosome-capturing Ab-conjugated siRNAs with branched Arg linkers can be effectively delivered into MM cells via uptake of exosomes by parental cells. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic cancers.

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.