Hematological Cancer Cell Research Articles

CD70 CAR NK Cells in the Treatment of Multiple Myeloma

Despite the high response rates of chimeric antigen receptor (CAR) - T cells targeting the B-cell maturation antigen (BCMA) for multiple myeloma (MM), patients eventually still relapse. At this time there is no clear subsequent therapy. Furthermore, CAR T-cell therapy is associated with significant toxicities such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. CD70 is a normally transiently expressed on activated T-cells, B-cells, or dendritic cells, but have been found to be upregulated on many hematologic and solid cancer cells such as acute myeloid leukemia, triple negative breast cancer, and renal cell carcinoma. However, CD70 as a potential target for treatment of multiple myeloma, including patients who have progressed on BCMA targeted therapies, has not been fully investigated. We assessed bone marrow aspirates from MM patients by flow cytometry and demonstrated that CD70 was expressed on the majority of myeloma cells, and on average, relapsed myeloma patients had higher expression of CD70. In addition, using immunohistological staining, we found that CD70 was expressed on 10 of 10 patients who had progressed on BCMA targeted therapies (seven who had progressed on belantamab mafodotin, an antibody drug conjugate, three who had progressed on idecabtagene vicleucel, a BCMA CAR-T), suggesting that CD70 could be a viable target in patients who have failed BCMA targeted therapy. As natural killer (NK) cells are a promising off-the-shelf treatment with minimal toxicity as compared to CAR-T cells, we generated CD70-targeting CAR NK cells for the treatment of MM. We found in the chromium cytotoxicity assay that CD70 CAR NK cells had higher killing as compared to non-transduced (NT) NK cells at multiple effector-to-target ratios against MM1s, a myeloma cell line confirmed to express CD70. There was no difference in killing when co-cultured with H929, a myeloma cell line lacking CD70 expression, demonstrating the specificity of the CD70 CAR NK cell. The enhanced killing was also confirmed in an incucyte assay in which the CD70 CAR NK cells cleared tumor faster than NT NK, even in MM1s in which BCMA was knocked out using CRISPR (simulating BCMA antigen loss). When tested in a myeloma mouse model, the CD70 CAR NK exhibited greater tumor control as shown in Figure 1, when compared to NT NK, which lead to increased survival of the mice. Similar results were seen in a mouse model using BCMA KO myeloma cells. In summary, we were able to demonstrate that CD70 can be a feasible target for the treatment of multiple myeloma, including in patients who have failed BCMA targeted therapy. Based on these results, we have initiated a Phase I/II clinical trial (NCT05092451) that is currently recruiting.

A Novel and Potent EZH1/2 Dual Inhibitor, HM97662 Demonstrated a Wide Spectrum of Therapeutic Potential for Hematological Malignancies

Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homolog 2 (EZH2) and its homolog EZH1 are catalytic components of polycomb repressive complex 2 (PRC2), which tri-methylate histone H3 at lysine 27 (H3K27me3) to repress transcription of their target genes. Hyperinduction of H3K27me3 mediated by EZH2 overexpression or EZH2 gain-of-function (GOF) mutation (e.g. Y641, A677, and A687) has been associated with the progression of hematological malignancies including B-cell lymphoma, T-cell lymphoma, and multiple myeloma. Hence, it has been suggested that suppressing PRC2 activity by EZH2 inhibition is a potential therapeutic target for hematological malignancies. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. Moreover, EZH1 directly binds to chromatin and modulates its condensation. Therefore, dual inhibition of EZH1 and EZH2 can give better effect than EZH2 inhibition alone in blocking PRC2 function as an anti-cancer therapy. Herein, we presented a novel EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of wild-type EZH1 as well as wild-type and GOF mutant EZH2 at nanomolar concentrations. SPR analysis of HM97662 on trimeric complexes (EZH1/EZH2, SUZ12, and EED) demonstrated that HM97662 binds both EZH1- and EZH2-PRC2 complexes. HM97662 potently repressed tri-methylation of H3K27 in KARPAS 422 DLBCL, HH PTCL, and MM.1S MM cell lines. Furthermore, HM97662 showed broader and stronger activities than EZH2 selective inhibitors in long-term proliferation assay against a variety of hematological cancer cell lines. HM97662 induced the differentiation of DLBCL to plasma cells with the increment of cell lineage specific markers (e.g. PRDM1 and CD38), and caused cell cycle G0/G1 arrest and apoptosis in KARPAS 422 DLBCL cells harboring EZH2 Y641N GOF mutation. Additionally, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH PTCL cell line. Two cell cycle repressors, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were significantly increased by HM97662. We further conducted a chromatin accessibility assay and identified that the chromatin structures of that genes were loosened and highly transcribed after the treatment of HM97662. As a result, HM97662 showed potent antitumor activity in various hematological cell lines xenograft mouse models including B-cell lymphoma, T-cell lymphoma, and multiple myeloma (e.g. KARPAS 422, HuT-102, and MM.1S). Once daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to the known EZH2 selective or EZH1/2 dual inhibitor. In conclusion, the present preclinical studies demonstrated that HM97662, an EZH1/2 dual inhibitor, had a promising and wide spectrum of therapeutic potential for hematological malignancies. It is urgent to assess the effectiveness of HM97662 in further clinical trials.

Paracrine Factors Released from Tonsil-Derived Mesenchymal Stem Cells Inhibit Proliferation of Hematological Cancer Cells Under Hyperthermia in Co-culture Model.

Mesenchymal stem cells (MSCs) are promising biological therapeutic candidates in cancer treatment. As a source of MSCs, palatine tonsil tissue isone of the secondary lymphoid organs that form an essential part of the immune system, and the relation betweenthe secondary lymphoid organs and cancer progression leads us to investigate the effect of tonsil-derived MSCs (T-MSC) on cancer treatment. We aimed to determine the anti-tumoral effects of T-MSCs cultured at the febrile temperature (40°C) on hematological cancer cell lines. The co-culture of cancer cells with T-MSCs was carried out under fever and normal culture conditions, and then the cell viability was determined by cell counting. In addition, apoptosis rate and cell cycle arrest were determined by flow cytometry. We confirmed the apoptotic effect of T-MSC co-culture at the transcriptional level by using real-time polymerase chain reaction (RT-PCR). We found that co-culture of cancer cells with T-MSCs significantly decreased the viable cell number under the febrile and normal culture conditions. Besides, the T-MSC co-culture induced apoptosis on K562 and MOLT-4 cells and induced the cell cycle arrest at the G2/M phase on MOLT-4 cells. The apoptotic effect of T-MSC co-culture under febrile stimulation was confirmed at thetranscriptional level. Our study has highlighted the anti-tumoral effect of the cellular interaction between the T-MSCs and human hematological cancer cells during in vitro co-culture under hyperthermia.

Investigation of mitochondrial oxidative phosforylation enzymes in hematological cancers

It is known that cancerous cells are different from normal cells in their structure and functions. It is aimed to determine whether there is a change in the activities of mitochondrial oxidative phosphorylation (OXPOS) respiratory enzymes, the main energy production site of cells, in various haematological cancers and to use the obtained data for therapeutic purposes. In this study, leukocyte mitochondria were isolated from blood samples obtained from patients with non-hodgkin lymphoma (NHL), hodgkin lemphoma (HL), chronic lymphocytic leukaemia (CLL), acute myelogenic leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML), multiple myeloma and myelodysplastic syndrome (MDS) and controls. The enzyme activities of oxidative phosphorylation complexes I, II and IV were measured by spectrophotometric method. Enzyme activity results were statistically evaluated according to various criteria. Enzyme activities were evaluated according to gender, chemotherapy or not and disease groups. As a result of these statistical evaluations, no correlation was found between the type of haematological cancer and gender (p>0.05). The difference in mitochondrial oxidative complex enzyme activities between patients receiving chemotherapy and those not receiving chemotherapy was insignificant (p>0.05). When the enzyme activities of the control group and all haematological cancer cells studied were compared, the difference was found to be statistically significant (p<0.05). As a result, changes in the activities of OXPOS respiratory enzymes were found in various haematological cancers. The large number of therapies that specifically target mitochondria, the altered metabolic functions and dynamics of mitochondria in cancer cells make it a target for cancer therapeutics.

Discovery of a non-covalent ligand for Rpn-13, a therapeutic target for hematological cancers

The ubiquitin–proteasome system serves as the major proteolytic degradation pathway in eukaryotic cells. Many inhibitors that covalently bind to the proteasome’s active sites have been developed for hematological cancers, but resistance can arise in patients. To overcome limitations of active–site proteasome inhibitors, we and others have focused on developing ligands that target subunits on the 19S regulatory particle (19S RP). One such 19S RP subunit, Rpn-13, is a ubiquitin receptor required for hematological cancers to rapidly degrade proteins to avoid apoptosis. Reported Rpn-13 inhibitors covalently bind to the Rpn-13′s Pru domain and have been effective anti-hematological cancer agents. Here, we describe the discovery of TCL-1, a non-covalent binder to the Pru domain. Optimization of TCL-1′s carboxylate group to an ester increases its cytotoxicity in hematological cancer cell lines. Altogether, our data provides a new scaffold for future medicinal chemistry optimization to target Rpn-13 therapeutically.

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells

Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

Bacterial Expression of TMTP1-Fused L-Asparaginase for Targeting Leukemia and Metastatic Tumor Cells

L-asparaginase is recognized as a first-line anticancer drug for acute lymphoblastic leukemia (ALL); however, low-substrate specificity and exhibiting glutaminase activity cause various off-target toxicities on normal cells. In the following study, we functionalized wild-type asparaginase with the TMTP1 targeting peptide which specifically targets a variety of hematological and metastatic cancer cells. The peptide sequence was genetically added to the N-terminal end of the asparaginase using the restriction endonuclease-free cloning method. Wild-type and engineered asparaginases were expressed in E. coli and purified by Nickel affinity chromatography column. The in vitro activity of both types of enzymes was evaluated by Nessler’s method. The sequencing results showed that the TMTP1 sequence was added in the correct frame to the asparaginase. Wild-type and TMTP1-fused asparaginases were produced in a soluble state with the specific activity of 172 U/mg and 153 U/mg, respectively. The evidence from this study suggests that TMTP1-fused asparaginase could preserve its solubility and activity compared to the wild-type species and can be proposed for future research in anticancer therapies.

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.

LP-284, a small molecule acylfulvene, exerts potent antitumor activity in preclinical non-Hodgkin's lymphoma models and in cells deficient in DNA damage repair

Despite advances in therapies treating non-Hodgkin's lymphoma (NHL), 20~40% of patients experience relapsed or refractory disease. While solid tumors with homologous recombination deficiencies have been successfully targeted with synthetic lethal agents such as poly-ADP ribose polymerase (PARP) inhibitors, such synthetic lethality strategy has not yet been approved to treat patients with NHL. Here we investigated the mechanism of action (MoA) and therapeutic potential of a new-generation acylfulvene compound, LP-284, in both in vitro and in vivo NHL models. One of LP-284's MoA includes inducing the repair of double-strand DNA break (DSB). We found that LP-284 exerts nanomolar potency in a panel of hematological cancer cell lines including fifteen NHL cell lines. In vivo, LP-284 treatment prolongs the survival of mantle cell lymphoma (MCL) cell line JeKo-1 derived xenograft mice by two-fold and shows increased efficacy over bortezomib and ibrutinib. In addition, LP-284 is capable of inhibiting tumor growth of JeKo-1 xenografts that are refractory to bortezomib or ibrutinib. We further showed that LP-284 is particularly lethal in cells with deficient DNA damage response and repair, a targetable vulnerability in NHL.

A first-in-human, open-label, phase I trial of daily oral PCLX-001, an NMT inhibitor, in patients with relapsed/refractory B-cell lymphomas and advanced solid tumors.

e15094 Background: Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, regulates multiple membrane-bound signal transduction pathways driving cancer cell biology. This modification is catalyzed by two N-myristoyltransferases (NMT), NMT1 and NMT2. Aberrant NMT expression has been identified in cancer cells and inhibition of myristoylation represents a novel anticancer treatment strategy. PCLX-001 is an oral, highly bioavailable, small molecule NMT inhibitor with strong affinity for both NMT1 and NMT2 proteins (IC50 of 5nM and 8nM, respectively). In vitro, hematologic cancer cell lines were exquisitely sensitive to PCLX-001. PCLX-001 regressed subcutaneous tumors in xenograft models derived from lymphoma cell lines, as well as in a refractory DLBCL patient derived xenograft model. PCLX-001 also demonstrated anticancer activity in multiple solid tumor xenograft models. Based on pre-clinical data, we hypothesized that PCLX-001 would be safe, tolerable, and active as a novel oral cancer therapeutic. Methods: Patients with relapsed/refractory B-cell lymphomas and advanced solid tumors are being enrolled in a multicenter, open label, standard phase I dose escalation design to determine feasibility, maximum tolerated dose (MTD), and pharmacokinetics of PCLX-001 monotherapy. Seven dose levels are being studied: 20 mg, 40 mg, 70 mg, 100 mg, 140 mg, 200 mg, and 280 mg. Dose limiting toxicity (DLT) is defined as: grade (Gr) 4 platelets, Gr 3 platelets with bleeding, Gr 4 ANC ≥ 7 days, febrile neutropenia, or clinically significant > Gr 3 non-hematologic toxicity. Results: Eighteen evaluable patients (3 relapsed B-cell lymphoma; 15 advanced solid tumor) have been accrued through dose level six (200 mg cohort) without DLT. No attributable Gr 3 or 4 toxicities have been identified. Noncompartmental pharmacokinetic (PK) analyses demonstrate rapid oral absorption, a terminal half-life of approximately 10 hours, rapid achievement of steady state, and no induction of metabolism. PCLX-001 trough plasma concentrations at higher dose cohorts exceed the EC90 required to inhibit cultured cancer cells. Conclusions: PCLX-001 is safe and well tolerated up to the 200 mg daily dose. Our results support the ongoing development of PCLX-001 as an oral daily therapy for the treatment of patients with cancer. Updated study results will be presented, summarizing the safety, MTD, and PK outcomes in PCLX-001 treated patients. Preliminary clinical activity will be determined in 20 patient dose expansion cohorts of relapsed/refractory B-cell lymphomas, and selected advanced solid tumors following the identification of PCLX-001 MTD. Clinical trial information: NCT04836195 .

Profiling of Natural Killer Interactions With Cancer Cells Using Mass Cytometry

We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors’ peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.

Abstract 1662: Understanding the sensitivity of cancer cells to myristoylation inhibitors for oncology applications

Abstract PCLX-001 is a first-in-kind drug that targets a protein modification essential for membrane anchorage and signalling, and has been under human clinical trial evaluation for the treatment of lymphoma and solid malignancies for over a year. In human, over 600 proteoforms are modified with the fatty acid myristate by two N-myristoyltransferases: NMT1 and NMT2. These include many proto-oncogenic proteins (e.g. Src-family kinases and c-Abl) and metabolic regulators (e.g. AMPK β subunit). Thus, we validated the potential of NMTs as oncology targets on 300 cancer cell lines and uncovered its potential in hematological cancers. The multiple substrates of NMTs complicated the elucidation of a clear mechanism of action of the pan-NMT inhibitor PCLX-001. To circumvent this, we evaluated the individual roles of NMT1 and NMT2 in tumours as well as their relative contributions, including those of their substrates, to PCLX-001 sensitivity. Analysis of TCGA and CCLE databases revealed that NMT2 expression levels vary drastically (7 log2(TPM+1)) while those of NMT1 vary slightly (3 log2(TPM+1)). Hematological cancers comprised the vast majority of the lowest NMT2 expressing cells but lower NMT2 levels were also observed in solid tumour cell lines (e.g. breast & ovary). NMT2 levels were also lower in cancer versus normal corresponding tissues. We confirmed that NMT2 expression is epigenetically repressed at a CpG island found in the 5´end of the NMT2 gene thereby explaining the reduction in NMT2 expression. Since most hematological cancer cells are NMT2-deficient, we think that by targeting the remaining NMT1, PCLX-001 selectively kills these cells in a manner reminiscent of synthetic lethality, thereby sparing normal human cells with two NMTs. Therefore, identifying substrates specific to NMT1 in NMT2-deficient cancer cells became of the utmost importance to understand which NMT1 protein substrates were critical for survival. Using differential proteomics in the CRISPR/Cas9 NMT1 KO HAP1 near-haploid cell line derived from the chronic myelogenous leukemia versus WT HAP1 cells, we surprisingly found that few signaling proteins were downregulated, rather, 36 mitochondrial protein levels were decreased and 18 of these belonged to respiratory complex I. Myristoylated mitochondrial protein NDUFAF4, a major complex I assembly factor, was the most downregulated and PCLX-001 reduced its levels leading to decreased complex I activity. This seemingly ordinary observation could have transformational implications for cancer treatment since oxidative phosphorylation is essential for both cancer stem cell survival and metastasis. In addition, gene set enrichment analyses performed on RNASeq data obtained from 1200 cell lines treated with myristoylation inhibitors allowed us to derive an NMT inhibition sensitivity gene set that could be used to identify patients which would most benefit from NMT inhibition therapy. Citation Format: Erwan Beauchamp, Chistopher Cromwell, Eman Moussa, Aishwarya Iyer, Megan Yap, Rony Pain, Jay Gamma, Olivier Julien, Basil Hubbard, Luc Berthiaume. Understanding the sensitivity of cancer cells to myristoylation inhibitors for oncology applications [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 1662.

Abstract 2934: BYON4228, an antagonistic SIRPα mAb with a unique and favorable preclinical profile compared to three comparator SIRPα mAbs

Abstract A growing body of data from early clinical studies supports that targeting the myeloid immune checkpoint CD47-SIRPα represents a successful strategy towards establishing effective new therapies for cancer patients. Initial studies focused on CD47 blockers and more recently also SIRPα antagonistic mAbs entered clinical studies. The latter approach is thought to be more advantageous since SIRPα expression is primarily myeloid restricted whereas CD47 is broadly expressed and -besides SIRPα- binds other ligands as well.We generated BYON4228, an antibody directed against the two existing allelic variants of human SIRPα that does not recognize the closely related T-cell expressed SIRPγ. Functional studies demonstrate that BYON4228 potentiates both macrophage- and neutrophil-mediated elimination of hematologic and solid cancer cells in vitro in the presence of a variety of tumor targeting antibodies, including rituximab, daratumumab, trastuzumab and cetuximab. In vivo, BYON4228 increases the anti-tumor activity of rituximab in a B-cell Raji xenograft model in mice and shows a favorable safety profile in cynomolgus monkeys.To position BYON4228 in the emerging field of myeloid checkpoint inhibitors, head-to-head comparison studies were performed. For these studies, previously described anti-SIRPα mAbs HEFLB, 1H9, and SIRPAB-11 were generated in house that in all probability are representative for three clinical stage SIRPα mAbs. These comparative studies focused on principal characteristics of this class of mAbs that are anticipated to affect their clinical profile, namely 1) recognition of both common allelic forms of SIRPα which determines the broadness of its clinical applicability; 2) binding to SIRPγ on T-lymphocytes which is reported to affect T-cell extravasation and activation and thereby the adaptive immune response; 3) functionality of the Fc-region of the mAb which can mediate depletion of SIRPα-positive host myeloid immune effector cells thereby compromising host defense and undermining the effect of immunotherapy. The comparative studies show that in contrast to HEFLB, BYON4228 recognizes both common allelic forms of SIRPα. Furthermore, lack of binding to T-cell expressed SIRPγ differentiates BYON4228 from 1H9 and SIRPAB-11. Finally, with its impaired Fc-region BYON4228 does not deplete SIRPα+ myeloid cells, in contrast to SIRPAB-11.Thus, BYON4228 differentiates from all three comparator SIRPα mAbs based on at least one fundamental characteristic, thereby underlining its potential to become a best-in-class drug. We therefore endorse its further development for which clinical studies are planned to start in 2023. Citation Format: Mary van Helden, Seline Zwarthoff, Roel Arends, Inge Reinieren-Beeren, Marc Parade, Lilian Driessen-Engels, Karin de Laat-Arts, Desiree Damming, Ellen Santegoeds-Lenssen, Daphne van Kuppeveld, Imke Lodewijks, Ellen Mattaar, Hugo Olsman, Hanke Matlung, Katka Franke, Marloes Stokman, Benny de Wit, Dirk Glaudemans, Danielle van Wijk, Lonnie Joosten Stoffels, Jan Schouten, Paul Boersema, Monique van der Vleuten, Jorien Sanderink, Wendy Kappers, Diels van den Dobbelsteen, Marco Timmers, Ruud Ubink, Gerard Rouwendal, Gijs Verheijden, Miranda M.C. van der Lee, Wim Dokter, Timo van den Berg. BYON4228, an antagonistic SIRPα mAb with a unique and favorable preclinical profile compared to three comparator SIRPα mAbs [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 2934.

Abstract 1652: A novel reversible inhibitor of XPO1 with potent efficacy in multiple preclinical mouse models

Abstract Exportin 1 (XPO1/CRM1) is a key nuclear transport receptor (karyopherin) responsible for the export of different cargo proteins out of the cell’s nucleus into the cytoplasm. Its correct function is essential for cellular homeostasis, however normal XPO1 function is often disrupted in malignant cells. XPO1 is overexpressed in solid and hematological tumors and higher XPO1 expression is often associated with poorer prognosis. XPO1-regulated cargo proteins include tumor suppressor proteins and cell cycle regulators that can be involved in tumorigenesis. For example, aberrant XPO1 function may lead to cytoplasmic mislocalization of tumor suppressor proteins which results in their functional inactivation and hence may cause tumorigenesis. Inhibition of XPO1 function will restore their proper subcellular localization and function and cause tumor regression. Although XPO1 has a central role in cellular homeostasis, it is a good target for cancer therapy, as illustrated by the clinical success of the selective inhibitor of nuclear export (SINE) selinexor. Selinexor is the first and currently the only XPO1 inhibitor clinically approved. It is applied for the treatment of patients with multiple myeloma after at least one prior treatment, and for the treatment of diffuse large B cell lymphoma. Selinexor covalently binds to XPO1. The second generation SINE eltanexor is also a covalent XPO1 inhibitor but has only minimal brain penetration and consequently lower toxicity in preclinical studies. Here, we describe a novel chemical class of reversible XPO1 inhibitors with high brain penetration but with good tolerability allowing frequent dosing in preclinical models. The lead compound, FR-027, potently inhibits XPO1 function (EC50 69 ± 10 nM) and shows potent cancer cell growth inhibition in vitro of both hematological and solid cancer cell lines (EC50 50-950 nM). FR-027 is a reversible inhibitor of XPO1 and unlike many other XPO1 inhibitors it does not induce XPO1 protein degradation; this may contribute to an increased tolerability. Indeed, while it shows high brain penetration, FR-027 allows frequent dosing in mice with good tolerability (body weight). It is orally bioavailable and demonstrates strong anti-leukemic efficacy in an aggressive MOLT-4 xenograft model. Importantly, it shows potent efficacy in both an orthotopic U87 MG brain tumor xenograft model and a metastatic syngeneic ID8-fLuc ovarian cancer model with significant survival benefit as monotherapy. Altogether, these results demonstrate that FR-027 is a novel, reversible XPO1 inhibitor with important molecular and pharmacological characteristics that warrant further clinical development. Citation Format: Janne Van Hauwenhuyse, Felien Reniers, Leentje Persoons, Sam Noppen, Eline Boel, Els Vanstreels, Ann Vankerckhoven, Bert Kwanten, An Coosemans, Guy Van den Mooter, Wim Dehaen, Dirk Daelemans. A novel reversible inhibitor of XPO1 with potent efficacy in multiple preclinical mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1652.

BYON4228 is a pan-allelic antagonistic SIRPα antibody that potentiates destruction of antibody-opsonized tumor cells and lacks binding to SIRPγ on T cells

BackgroundPreclinical studies have firmly established the CD47-signal-regulatory protein (SIRP)α axis as a myeloid immune checkpoint in cancer, and this is corroborated by available evidence from the first clinical studies with...

Development of Curcumin and Piperine-Loaded Bio-Active Self-Nanoemulsifying Drugs and Investigation of Their Bioactivity in Zebrafish Embryos and Human Hematological Cancer Cell Lines.

Curcumin (CUR) and piperine (PP) are bioactive compounds with prominent pharmacological activities that have been investigated for the treatment of various diseases. The aim of the present study is to develop Bio-SNEDDS for CUR and PP as a combined delivery system for cancer therapy. CUR and PP loaded Bio-SNEDDSs with varying compositions of bioactive lipid oils, surfactants, and cosolvents were prepared at room temperature. Bio-SNEDDSs were characterized using a Zetasizer Nano particle size analyzer and further examined by transmission electron microscopy (TEM) for morphology. The in vivo toxicity of the preparations of Bio-SNEDDS was investigated in wild-type zebrafish embryos and cytotoxicity in THP-1 (human leukemia monocytic cells), Jurkat (human T lymphocyte cells) and HUVEC (non-cancerous normal) cells. Bio-SNEDDSs were successfully developed with black seed oil, Imwitor 988, Transcutol P and Cremophor RH40 at a ratio of 20/20/10/50 (%w/w). The droplet size, polydispersity index and zeta potential of the optimized Bio-SNEDDS were found to be 42.13 nm, 0.59, and -19.30 mV, respectively. Bio-SNEDDS showed a spherical structure evident by TEM analysis. The results showed that Bio-SNEDDS did not induce toxicity in zebrafish embryos at concentrations between 0.40 and 30.00 μg/mL. In TG (fli1: EGFP) embryos treated with Bio-SNEDDS, there was no change in the blood vessel structure. The O-dianisidine staining of Bio-SNEDDS treated embryos at 48 h post-fertilization also showed a significant reduction in the number of blood cells compared to mock (DMSO 0.1% V/V) treated embryos. Bio-SNEDDS induced significant levels of cytotoxicity in the hematological cell lines THP-1 and Jurkat, while low toxicity in normal HUVEC cell lines was observed with IC50 values of 18.63±0.23 μg/mL, 26.03 ± 1.5 μg/mL and 17.52 ± 0.22 μg/mL, respectively. Bio-SNEDDS exhibited enhanced anticancer activity and could thus be an important new pharmaceutical formulation to treat leukemia.

Synthesis and structure-activity relationship of new nicotinamide phosphoribosyltransferase inhibitors with antitumor activity on solid and haematological cancer

Cancer cells are highly dependent on Nicotinamide phosphoribosyltransferase (NAMPT) activity for proliferation, therefore NAMPT represents an interesting target for the development of anti-cancer drugs. Several compounds, such as FK866 and CHS828, were identified as potent NAMPT inhibitors with strong anti-cancer activity, although none of them reached the late stages of clinical trials. We present herein the preparation of three libraries of new inhibitors containing (pyridin-3-yl)triazole, (pyridin-3-yl)thiourea and (pyridin-3/4-yl)cyanoguanidine as cap/connecting unit and a furyl group at the tail position of the compound. Antiproliferative activity in vitro was evaluated on a panel of solid and haematological cancer cell lines and most of the synthesized compounds showed nanomolar or sub-nanomolar cytotoxic activity in MiaPaCa-2 (pancreatic cancer), ML2 (acute myeloid leukemia), JRKT (acute lymphobalistic leukemia), NMLW (Burkitt lymphoma), RPMI8226 (multiple myeloma) and NB4 (acute myeloid leukemia), with lower IC50 values than those reported for FK866. Notably, compounds 35a, 39a and 47 showed cytotoxic activity against ML2 with IC50 = 18, 46 and 49 pM, and IC50 towards MiaPaCa-2 of 0.005, 0.455 and 2.81 nM, respectively. Moreover, their role on the NAD+ synthetic pathway was demonstrated by the NAMPT inhibition assay. Finally, the intracellular NAD+ depletion was confirmed in vitro to induced ROS accumulation that cause a time-dependent mitochondrial membrane depolarization, leading to ATP loss and cell death.

A Novel Peptide that Disrupts the Lck-IP3R Protein-Protein Interaction Induces Widespread Cell Death in Leukemia and Lymphoma.

There is increasing evidence that the T-cell protein, Lck, is involved in the pathogenesis of chronic lymphocytic leukemia (CLL) as well as other leukemias and lymphomas. We previously discovered that Lck binds to domain 5 of inositol 1,4,5-trisphosphate receptors (IP3R) to regulate Ca2+ homeostasis. Using bioinformatics, we targeted a region within domain 5 of IP3R-1 predicted to facilitate protein-protein interactions (PPIs). We generated a synthetic 21 amino acid peptide, KKRMDLVLELKNNASKLLLAI, which constitutes a domain 5 sub-domain (D5SD) of IP3R-1 that specifically binds Lck via its SH2 domain. With the addition of an HIV-TAT sequence to enable cell permeability of D5SD peptide, we observed wide-spread, Ca2+-dependent, cell killing of hematological cancer cells when the Lck-IP3R PPI was disrupted by TAT-D5SD. All cell lines and primary cells were sensitive to D5SD peptide, but malignant T-cells were less sensitive compared with B-cell or myeloid malignancies. Mining of RNA-seq data showed that LCK was expressed in primary diffuse large B-cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML). In fact, LCK shows a similar pattern of expression as many well-characterized AML oncogenes and is part of a protein interactome that includes FLT3-ITD, Notch-1, and Kit. Consistent with these findings, our data suggest that the Lck-IP3R PPI may protect malignant hematopoietic cells from death. Importantly, TAT-D5SD showed no cytotoxicity in three different non-hematopoietic cell lines; thus its ability to induce cell death appears specific to hematopoietic cells. Together, these data show that a peptide designed to disrupt the Lck-IP3R PPI has a wide range of pre-clinical activity in leukemia and lymphoma.

PCNA regulates primary metabolism by scaffolding metabolic enzymes

The essential roles of proliferating cell nuclear antigen (PCNA) as a scaffold protein in DNA replication and repair are well established, while its cytosolic roles are less explored. Two metabolic enzymes, alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), both contain PCNA interacting motifs. Mutation of the PCNA interacting motif APIM in ENO1 (F423A) impaired its binding to PCNA and resulted in reduced cellular levels of ENO1 protein, reduced growth rate, reduced glucose consumption, and reduced activation of AKT. Metabolome and signalome analysis reveal large consequences of impairing the direct interaction between PCNA and ENO1. Metabolites above ENO1 in glycolysis accumulated while lower glycolytic and TCA cycle metabolite pools decreased in the APIM-mutated cells; however, their overall energetic status were similar to parental cells. Treating haematological cancer cells or activated primary monocytes with a PCNA targeting peptide drug containing APIM (ATX-101) also lead to a metabolic shift characterized by reduced glycolytic rate. In addition, we show that ATX-101 treatments reduced the ENO1 - PCNA interaction, the ENO1, GAPDH and 6PGD protein levels, as well as the 6PGD activity. Here we report for the first time that PCNA acts as a scaffold for metabolic enzymes, and thereby act as a direct regulator of primary metabolism.

Novel pyridine-containing histone deacetylase inhibitors strongly arrest proliferation, induce apoptosis and modulate miRNAs in cancer cells

After over 30 years of research, the development of HDAC inhibitors led to five FDA/Chinese FDA-approved drugs and many others under clinical or preclinical investigation to treat cancer and non-cancer diseases. Herein, based on our recent development of pyridine-based isomers as HDAC inhibitors, we report a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2′-aminoanilides 5–8 as anticancer agents. The hydroxamate 5d proved to be quite HDAC3/6-selective exhibiting IC50 values of 80 and 11 nM, respectively, whereas the congener 5e behaved as inhibitor of HDAC1-3, −6, −8, and −10 (class I/IIb-selective inhibitor) at nanomolar level. Compound 5e provided a huge antiproliferative activity (nanomolar IC50 values) against both haematological and solid cancer cell lines. In leukaemia U937 cells, the hydroxamate 5d and the 2′-aminoanilide 8f induced remarkable cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, showing a stronger effect with respect to SAHA and MS-275 used as reference compounds. In U937 cells, the highest dose- and time-dependent cytodifferentiation was obtained by the 2′-aminoanilide 8d (up to 35% of CD11c positive/propidium iodide negative cells at 5 μM for 48 h). The same 8d and the hydroxamates 5d and 5e were the most effective in inducing p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f increased mRNA expression of p21, BAX and BAK, downregulated cyclin D1 and BCL-2 and modulated pro- and anti-apoptotic microRNAs towards apoptosis induction. Finally, 5e strongly arrested proliferation in nine different haematological cancer cell lines, with dual-digit nanomolar potency towards MV4-11, Kasumi-1, and NB4, being more potent than mocetinostat, used as reference drug.