Parental Cell Line Research Articles

Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line.

Antiviral vaccines for pig diseases are essential to prevent epidemic outbreaks. However, their production is often hindered by inefficient manufacturing processes that yield lower quantities of the vaccine. To accelerate the progress of various areas of bioproduction, we have considered the necessity of enhancing viral replication efficiency by optimizing ST (swine testicular) cell lines that are commonly utilized in virus manufacturing. CRISPR/Cas9 gene-editing technology were utilized to create IRF3 or IRF7 knockout cell lines that facilitate high-titer viral stock production. Compared to the parental cell lines, the ST IRF3/7 KO cell line displayed a compromised antiviral response to a panel of viruses (Porcine epidemic diarrhea virus, Senecavirus A, Parainfluenza virus 5, and Getah virus), as evidenced by decreased expression of interferon and certain antiviral factors. The inhibition of these responses led to heightened viral replication and increased cytopathic effects, ultimately promoting apoptosis. As a result, the development of these cell lines offers a more efficient approach for biopharmaceutical companies to boost their virus production and reduce associated costs.

Identification of multiple organ metastasis-associated hub mRNA/miRNA signatures in non-small cell lung cancer

Metastasis remains major cause of treatment failure in non-small cell lung cancer (NSCLC). A comprehensive characterization of the transcriptomic landscape of NSCLC-cells with organ-specific metastatic potentials would advance our understanding of NSCLC metastasis process. In this study, we established NSCLC bone-metastatic (BoM), brain-metastatic (BrM), and lymph-metastatic (LnM) cells by an in vivo spontaneous metastatic model. Subsequently, by analyzing the entire transcriptomic profiles of BoM, BrM, LnM, LuM, in comparison with their parental cell line L9981, we identified miR-660-5p as a key driver that is associated with NSCLC progression and distant metastasis, potentially through its targeting of LIMCH1, SMARCA5 and TPP2. In addition, a six-gene signature (ADRB2, DPYSL2, IL7R, LIMCH1, PIK3R1, and SOX2) was subsequently established to predict NSCLC metastasis based on differentially expressed genes, three of which (DPYSL2, PIK3R1, LIMCH1) along with the transcriptional factors RB1 and TP63, were ultimately validated by experiments. Taken together, aberrant gene signature and miRNA can serve as biomarkers for predicting NSCLC distant metastasis, and targeting them could potentially contribute to the development of novel therapeutic strategies.

Abstract C163: KRAS alterations combined with TP53 mutations as novel synthetic lethal genomic lesions for PKMYT1 inhibition

Abstract Background: Membrane-associated tyrosine- and threonine-specific Cdc2-inhibitory kinase (PKMYT1) is a cell cycle regulatory kinase that inhibits CDK1/CyclinB activity, delaying entry into mitosis in tumor cells experiencing replication stress (RS). RS is frequently induced by genetic alterations that drive premature transition from G1 to S phase, promoting genome instability and creating a synthetic lethal (SL) relationship between these specific alterations and PKMYT1 inhibition. This relationship has been demonstrated preclinically and clinically with CCNE1 amplification and FBXW7 mutations, common alterations in ovarian and colorectal cancers, respectively. Oncogenic KRAS gain of function (GOF) mutations, combined with TP53 alterations, are bona fide drivers of RS. Here we investigate the relationship between KRAS/TP53 alterations and PKMYT1 inhibition, mediated by the first-in-class, potent and selective PKMYT1 inhibitor lunresertib (RP-6306), alone or in combination with RS-inducing agents. Methods: KRAS/TP53 double mutant isogenic cell pairs, across relevant tumor origins, were genetically engineered to express multiple KRAS GOF mutant codons and alleles (G12D, G12C, G12V, G12R, G13D and Q61H). Cell cycle distribution perturbations and RS were evaluated. Sensitivity to lunresertib alone or in combination with chemotherapeutic or targeted agents was assessed by cell growth assays. In vitro findings were further evaluated in KRAS/TP53 double mutant patient-derived xenografts (PDX). Results: Lunresertib inhibited the growth of isogenic KRAS/TP53 double mutant cells, with up to a 12-fold EC50 shift compared to control parental cells. Combination of lunresertib with RS-inducing chemotherapeutic agents (eg. gemcitabine), or the ATR inhibitor camonsertib (RP-3500) led to synergistic cell growth inhibition in KRAS/TP53 mutant cells at significantly lower concentrations than in wild type lines. Mechanistically, KRAS/TP53 isogenic cells showed elongated S-phase and increased cyclin B1 levels, phenocopying the effects of CCNE1 amplification. Lunresertib induced premature mitosis and DNA damage in KRAS/TP53 mutant cells, sparing parental cell lines. PDX efficacy studies demonstrated robust combination benefit in pancreatic, lung and colorectal settings, with durable tumor regressions and complete responses observed. Conclusion: GOF KRAS mutations, when combined with TP53 alterations, show a strong SL relationship with PKMYT1 inhibition, alone or in combination with RS-inducing antitumor agents. Importantly, the SL phenotype was observed across multiple tumor indications and KRAS mutant codons/alleles, suggesting a potential broad scope of clinical utility beyond approved KRAS inhibitors. The mechanism is similar to CCNE1 amplification, characterized by enhanced G1/S transition driving heightened dependence on PKMYT1 to inhibit CDK1/CyclinB. Taken together, our data form rationale for the therapeutic evaluation of PKMYT1 inhibitors in KRAS/TP53 altered tumors and uncover a potential new patient selection biomarker for lunresertib. Citation Format: Marc L. Hyer, Jimmy Fourtounis, David Gallo, Vivek Bhaskaran, Rino Stocco, Rosie Kryczka, Sai Save, Helen Burston, Olivier Nicolas, Stephen Morris, Anne Roulston, Jordan T. F. Young, Michal Zimmermann, C. Gary Marshall, Artur Veloso, Elia Aguado-Fraile. KRAS alterations combined with TP53 mutations as novel synthetic lethal genomic lesions for PKMYT1 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C163.

Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers

Abstract Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities. Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B173.

A STAT3 Degrader Demonstrates Pre-Clinical Efficacy in Venetoclax Resistant Acute Myeloid Leukemia

Introduction: Acute Myeloid leukemia (AML) is an aggressive hematological malignancy resulting from the transformation of immature myeloid progenitor cells followed by an uncontrolled clonal proliferation and accumulation of the transformed cells. AML is the most common myeloid malignancy in the elderly [1]. Venetoclax (Ven) is a selective inhibitor of the anti-apoptotic BCL2 protein. Ven is FDA approved in combination with hypomethylating agents (HMA's) or low dose cytarabine for the treatment of de-novo AML in patients > 75 years or those ineligible for standard induction therapies. Despite the recent FDA approval of HMA/Ven and other novel therapies, 5-year survival in newly diagnosed AML remain less than 30% and disease relapse is inevitable without a bridge to allogeneic stem cell transplantation [2]. Signal transducer and activator of transcription 3 (STAT3) belonging to the STAT family of transcription factors is well known to be inappropriately activated in several malignancies [3]. On stimulation by cytokines such as IL-6, STAT3 undergoes dimerization and phosphorylation. Phosphorylation at Tyr 705 leads to its translocation to the nucleus whereas phosphorylation at Ser 727 translocates it to the mitochondria [4]. Previous data from our lab demonstrated de-methylation and overexpression of STAT3 in MDS & AML stem cells, that is associated with an adverse prognosis [5]. We have also reported that STAT3 controls several important leukemic drivers such as the anti-apoptotic protein myeloid cell leukemia-1 (MCL1). MCL1 overexpression is the central mechanism of resistance to BCL2 inhibition (Ven) in AML [5]. While MCL1 is a well-known direct transcriptional target of STAT3, the role of STAT3 in venetoclax resistance (Ven-res) is unknown. Methods & Results: To understand the role of STAT3 in Venetoclax resistance (Ven-res), Ven-res AML cell lines (MOLM-13, MV-4-11) as well as a Ven-res large cell lymphoma cell line (SU-DHL-1) were generated, which exhibited increased levels of both total STAT3, phospho-STAT3 and its downstream effector, MCL1, when compared to their parental cell lines (Fig. 1A). Data from > 90 AML patients treated with prior Ven also showed high expression of total STAT3 along with enhanced phosphorylation for p Ser727 and p Tyr-705 STAT3, that strongly correlated with worse overall survival (OS) and reduced remission duration (RemDur) (p<0.05). A highly specific potent heterobifunctional degrader of STAT3 (degrader) resulted in a dose dependent and selective degradation of STAT3 in both parental and Ven-res hematological malignancy cell lines. Treatment with degrader showed a significant (>60%) decrease in p Tyr-705 as wellas p Ser-727 STAT3 levels in MOLM-13 parental as well as MOLM-13 Ven-res cells. STAT3 degradation also led to induction of apoptosis in both parental and Ven-res AML cell lines (p < 0.001). Importantly, colony assay of Ven-res AML patient samples showed effective degradation of STAT3 (>90%) together with the increased erythroid and myeloid differentiation (~ 2 fold) on treatment with degrader. Interestingly, no differentiation was observed in healthy samples, suggesting the specificity of the degrader to AML stem and progenitor cells. In patients with Ven-res AML, erythroid colony counts were seen to increase (~1.5 fold) with a concomitant decrease in myeloid colony counts (>2.5 fold), on treatment with degrader. This observation supports the clinical significance of our studies in AML patients with anemia. Although BH3 profiling of Ven-res MOLM-13 supports an increased dependency on MCL1, we have observed that treatment with degrader led to ~20% reduction in the MCL1 dependent mitochondrial depolarization. Additionally, cell derived xenograft (CDX) models of Ven-res showed significant reduction of p Tyr-705 STAT3(~60%), total STAT3 (>90%) and MCL1 (~70%), on treatment with STAT3 degrader - KT-333 (currently in an early phase clinical trial:NCT05225584), as early as week 2 (Fig 1B). Significant decrease in intracellular STAT3 in the spleen cells was also observed (p < 0.05), with ongoing survival analysis. Conclusion: Our study suggests that targeting STAT3 and the downstream MCL1 represents a novel and effective strategy for Ven-resistant AML patients in clinic, with strong mechanistic rationales that can spur further clinical development of STAT3 degraders especially given the significant side effects of direct MCL1 inhibitors.

Skp2 Inhibition Diminishes Essential Cell Survival Markers c-Maf and c-Myc, Enhancing Therapeutic Efficacy Against Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) presents challenges as a hematologic malignancy, with drug resistance leading to limited curative options and disease relapse despite advancements in therapy. Understanding the molecular mechanisms driving MM's development and progression is vital for identifying new treatment approaches. Clinical and experimental evidence supports the role of S-phase kinase-associated protein 2 (Skp2) in driving the progression of several cancers, including MM. We interrogated a public microarray dataset derived from clinical specimens of MM patients and observed a significantly higher expression of Skp2 within the relapsed patient cohort (N=28) compared to the newly diagnosed group (N=63) (P=0.0041). Additionally, we observed elevated Skp2 levels and decreased P27 Kip1 levels in drug-resistant MM cell lines (MM.1R and RPMI 8226.R5) compared to their parental cell lines (MM.1S and RPMI 8226). Hence, a comprehensive understanding of Skp2's molecular functions in drug-resistant MM becomes paramount for facilitating the development of precision-targeted interventions. We investigated Skp2's molecular interactions in MM by performing a targeted knockdown with siRNA in drug-resistant and sensitive cell lines (OPM1, RPMI.8226, MM.1S, and MM.1R). Immunoblot analysis showed reduced STAT3 activation and decreased levels of cell survival markers c-Maf and c-Myc. Treatment with 10uM of the small molecule inhibitor SkpinC1 also inhibited STAT3 activation and led to significant reductions in c-Maf and c-Myc protein levels compared to the vehicle-treated groups. These findings hold immense significance as c-Maf and c-Myc are intricately associated with MM progression and poorer outcomes, although there is limited information on their regulation by the Skp2/P27 axis. Functionally, treatment of the MM cell lines with increasing dosages of SkpinC1 showed a progressive decrease in MM cell line survival, with IC50 values spanning 5-15uM, signifying the broad applicability of the inhibitor among the tested cell lines. Moreover, flow cytometry-based analysis of the surviving cell population demonstrated that SkpinC1 based inhibition of the Skp2/P27 axis considerably decreased the percentage of proliferating M-phase cells, while elevating the proportion of quiescent and apoptotic cells relative to the control group. At the molecular level, targeted inhibition of the Skp2/P27 axis by SkpinC1 caused the accumulation of cell cycle inhibitors P27 Kip1, P21 (Cip1/Waf1), and P57 Kip2, activation of the tumor suppressive TP53 protein with a simultaneous decrease in phospho-MDM2 levels in addition to an increase in the levels of apoptotic markers cleaved PARP and Caspase 3. From a clinical perspective, we also probed the applicability of Skp2 inhibition using SkpinC1 as a potential strategy for managing therapeutic resistance in MM. We treated multiple MM cell lines (OPM1, RPMI.8226, MM.1S and MM.1R) using a combination of Lenalidomide (an immune modulator) or Bortezomib (a proteasomal inhibitor) with SkpinC1 and compared it with single agent treatments. Both Lenalidomide/SkpinC1 as well as Bortezomib/SkpinC1 showed a strong dose dependent decrease in the percentage of surviving cells relative to the single agent treatments, thereby highlighting the remarkable potential of targeting the Skp2/P27 axis for clinical therapy in MM. To gain deeper insights into the molecular roles of Skp2/P27 axis inhibition by SkpinC1, we subjected drug-resistant MM.1R cells to 10uM SkpinC1 treatment for 24 hours, followed by single-cell transcriptome sequencing. This approach illuminated considerable heterogeneity between the drug-treated and control cells, accompanied by global changes in various signaling pathways associated with cell survival. In conclusion, the inhibition of Skp2 in MM cells exerts a significant impact on crucial cell survival pathways, notably c-Maf and c-Myc, thereby promoting apoptosis and offering a promising therapeutic avenue to combat drug resistance in MM. The precise targeting of Skp2 using small molecule inhibitors, such as SkpinC1, demonstrates substantial therapeutic potential, enhancing the efficacy of combinatorial treatment approaches for more effective interventions in MM.

Debio 1562M, a Next Generation Antibody Drug Conjugate (ADC) Targeting CD37 for AML and MDS Treatment

CD37 is a trans-membrane protein specifically expressed on normal hematopoietic tissues such as B cells, neutrophils and macrophages, and has demonstrated roles in adhesion, cell migration (de Winde et al, 2018; Wee et al, 2015), survival and apoptotic signals (Lapalombella et al, 2012). Increased expression of CD37 has been observed in various hematological cancers such as lymphomas, Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML) (Beckwith et al, 2014; Deckert et al, 2013; Pereira et al, 2015; Yan et al, 2021). Higher expression of CD37 was also associated with poor patient outcome in AML (Zhang et al, 2020; Larkin et al, ASH 2018). Debio 1562M is a second-generation novel ADC directed against CD37 and including Debiopharm's proprietary Multilink cleavable linker-payload technology. Multilink improves manufacturing to achieve homogenous high drug antibody ratio while retaining excellent solubility as well as favorable biologic characteristics with preserved internalization kinetics on target cells. Debio 1562M is conjugated to 8 molecules of a tubulin-binding agent and despite cleavable linker that ensure effective intracellular delivery of toxic payload, we show sustained plasma stability in vitro and in vivo with no premature extracellular release. In this work, we report Debio 1562M specificity and potent antitumor activity in multiple AML models both in vitro and in vivo. First, in our in vitro cytotoxicity assay, Debio 1562M displayed nano- to picomolar anti-proliferative activity in both AML cell lines and primary samples. Out of 30 primary samples tested, 20 were sensitive to Debio 1562M with IC50 ranging from 1 to 10nM, this regardless of their mutation status, subtype or medical history. Interestingly, we observed that AML cells have high internalization capability and therefore relatively low levels of surface CD37 is sufficient for cytotoxic activity. The specificity of Debio 1562M was demonstrated by using 3 different CD37 knock out AML cell lines which were generated by CRISPR-Cas9 technology. Parental and CD37 KO cells were treated for 72h with a dose range of Debio 1562M and viability was measured (Figure 1). While Debio 1562M has an IC50 below 1nM in all 3 parental cell lines, its activity is reduced by more than 2 log in the KO cell lines. In vivo, in 3 different AML cell line xenograft mouse models, a single injection of Debio 1562M at 5mg/kg induced complete tumor regression and all mice remained alive at study termination, more than 60 days after treatment, except one mouse in one model. In a patient derived xenograft (PDX) model, the same dose of Debio 1562M significantly reduced tumor burden in blood and bone marrow with sustained activity 42 days after treatment (37% and 98% of blasts in blood and bone marrow respectively in control animals versus 0% and 3% in treated mice, pvalue<0.001). Furthermore, we evaluated the activity of Debio 1562M over venetoclax and azacytidine combination in the MV4-11 Luciferase xenografted mouse model. NCG mice were inoculated with 1x10 7 cells in the tail vein and randomization in groups of 8 mice was performed 14 days later. Mice received either one administration of Debio 1562M at 5mg/kg or a combination of venetoclax (QDx14 100mg/kg) and azacitidine (QDx5 3,5mg/kg). Complete regression of the tumor was observed in all mice after Debio 1562M administration and all mice survived at study termination, 75 days after treatment while none survived after 56 days when treated with venetoclax and azacitidine combination (Figure 2). The toxicology profile of Debio 1562M in mice showed expected clinical signs related to the payload's off target toxicity but with a significant safety margin. Interestingly, no peripheral neuropathy symptoms were detected, as commonly observed for this class of payload. Additionally, Multilink TM-payload construct alone was not associated with any signs of toxicity. On-target toxicity profile couldn't be evaluated as Debio 1562M isn't cross reactive with mouse CD37, however 1 st generation CD37 ADC has already demonstrated safety and tolerability in Phase 1 and 2 clinical trials.Overall, our data confirm the relevance of CD37 targeting for AML treatment. The excellent antitumor activity and tolerability of Debio 1562M in several pre-clinical models now pave the way for upcoming clinical development.

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

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

Biomarker Driven Strategies for Targeted Elimination of Hypoxia Adapted Lymphoma Cells

Large body of evidence suggests that hypoxia drives aggressive molecular features irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by widespread disease and frequent involvement of diverse hypoxic microenvironments (e.g., bone marrow, malignant effusions, large lymphoma masses). Impact of long-term hypoxia on the biology of lymphoma cells and its potential role in mediating drug resistance and disease relapses due to adaptive changes shaped by the hypoxic microenvironment remain only partially understood. In this study we analyzed impact of short- and long-term hypoxia on a panel of six lymphoma cell lines including diffuse large B-cell lymphoma (UPF4D, UPF8D), Burkitt lymphoma (Ramos, UPF9T), and mantle cell lymphoma (HBL2, MINO). Only 2 out of 8 tested lymphoma cell lines (Ramos, and HBL2) survived longer than 4 weeks under deep hypoxia (1% O2). The remaining tested lymphoma cell lines died by hypoxia-induced apoptosis (which could be inhibited by co-cultivation with pan-caspase inhibitor Z-VAD-FMK). The hypoxia-adapted (HA) Ramos and HBL2 cells had severely decreased proliferation rate accompanied by complex changes of the transcriptome, proteome, and metabolome. Seahorse analysis revealed significant inhibition of both oxidative phosphorylation and glycolytic pathways ( Figure 1). Of note, the whole-proteome profiling confirmed significant downregulation of proteins regulating oxidative phosphorylation, but significant upregulation of proteins regulating glycolysis. Sensitivity of HA cells to 2-deoxyglucose, an inhibitor of glycolysis was markedly increased ( Figure 1). The data suggest that under long-term deep hypoxia, lymphoma cells try to compensate for the decrease in ATP production from the oxidative phosphorylation process by boosting structural machinery of glycolysis, on which they became vitally dependent for survival. Despite the increased glycolytic machinary, however, the level of glycolysis decreases due to severe lack of oxygen. In our opinion, this is a clear representation of the Warburg effect, a hallmark of cancer based on the metabolic reprogramming of the cancer cells under hypoxia. Sensitivity of HA cells to a panel of the tested cytotoxic drugs (cytarabin, cisplatin, bortezomib) and targeted agents (venetoclax, S63545, A1155463, TRAIL, polatuzumab vedotin, IM-156, copanlisib) was either unchanged or increased (compared to sensitivity of the original cell lines cultured under normoxia). In contrast to so far published data, long-term hypoxia was not associated with acquired drug resistance in case of any of the tested agent. Of note, HA cells became significantly more sensitive to A1155463, an inhibitor of anti-apoptotic protein BCL-XL ( Figure 1). Unchanged levels of BCL-XL protein in HA cells and the respective normoxic cell lines (detected by western blotting) suggest that more complex mechanisms plausibly underlie the observed markedly increased sensitivity to A1155463. The whole-transcriptome and proteome analyses of both HA cell lines (compared to the parental cell lines cultured under normoxic conditions) detected among other complex changes signficant upregulation of P4HA1 (both mRNA, and protein), a prolyl hydroxylase involved in the stabilization of hypoxia-induced factor (HIF) 1 alpha, and putative oncogene associated with tumor aggressiveness. Transgenic (over)expression of P4HA1 in hypoxia-sensitive MINO cells effectively inhibited the hypoxia-induced apoptosis. Western blot analysis implemented on a panel of primary lymphoma cells revealed markedly higher expression of P4HA1 protein in the lymphoma cells obtained from malignant effusions (putative hypoxic microenvironment) compared to lymphoma cells obtained from leukemized blood (putative normoxic microenvironment). Our data suggest that P4HA1 positively impacts survival of lymphoma cells under hypoxia. In translation, P4HA1, BCL-XL, and structural proteins of the glycolytic pathway may represent novel drugable targets for more effective elimination of hypoxia-adapted lymphoma cells. Financial Support: Ministry of Health of the Czech Republic AZV NU23-03-00172, Grant Agency of the Czech Republic GA23-05377S, and National Institute for Cancer Research (EXCELES) LX22NPO5102.

Epigenetic Modifiers MMSET and EZH2 Physically Interact and Cooperate to Support Multiple Myeloma Pathophysiology

Multiple myeloma (MM) is a malignant B cell dyscrasia characterized by the accumulation of clonal plasma cells (PC) within the bone marrow (1). Genome sequencing studies have revealed great heterogeneity and genomic instability, a complex mutational landscape and a branching pattern of clonal evolution (2-4). Epigenetic factors are also involved in MM initiation, progression, and occurrence of chemoresistance (5). Among them, EZH2 and MMSET are associated with poor prognosis values and contribute to MM disease. EZH2 is the main catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and deposits H3K27me3 on chromatin (6), whereas MMSET, coded by the gene NSD2, which is the target oncogene of t(4;14) primary translocation, is a histone methyltransferase that methylates H3K36me2 (7). Using proteomics analysis of EZH2-immune precipitates, we identified a constitutive interaction between EZH2 and MMSET independently of t(4;14) translocation and mutational status of EZH2 in MM cells. Furthermore, MAK-683, an allosteric inhibitor of EZH2, disrupted this interaction, but not EPZ-6438, a catalytic inhibitor of EZH2, suggesting that MMSET-EZH2 interaction may be mediated by other PRC2 factors, but is independent of the PRC2 activity. Analysis of EZH2 expression levels and t(4;14) translocation status in 3 independent cohorts of MM patients (CoMMPass (N=674), UAMS-TT2 (N=345), Mtp-Daratumumab (N=51)) identified a worse overall survival of patients with high EZH2 expression combined with t(4;14) chromosomal translocation than patients with only one of these genetic events. We identified 355 differentially expressed genes in this “ EZH2 High t(4;14) +” subgroup compared to other patients. GSEA analysis of up-regulated genes in this subgroup identified a significant enrichment for genes involved in cell cycle, KRAS signaling, Cell-Cell signaling, Hypoxia and Epithelial-Mesenchymal transition. These results indicate that EZH2-MMSET interaction is of clinical interest in t(4;14) MM patients. To better understand the molecular relationship between EZH2 and MMSET, we generated MMSET-depleted clones by CRISPR-Cas9. MMSET depletion did not significantly alter cell proliferation and survival, but reduced the clonogenic capacity of MM cells compared to non-depleted cells. Moreover, after IL-6 or serum starvation, IL-6 and IGF-1 sensitivity was significantly affected in MMSET-depleted clones compared to the parental cell line, indicating that MMSET overexpression confers a cell growth advantage to MM cells under stress conditions and an independence of growth factors produced by bone marrow microenvironment Additionally, MMSET depletion induced changes in PRC2 nuclear localization in MM cells, a decrease of H3K36me3 and a concomitant increase in H3K27me3 levels, which altered the transcriptional program of cells showing, among others, a specific enrichment in genes up-regulated in t(4;14) MM subgroup of patients. Finally, we demonstrated that disrupting EZH2-MMSET interaction with MAK-683 may be of therapeutic interest, since MAK-683 combination with conventional drugs used in MM treatment, including melphalan and Panobinostat or BRD4 inhibitors, synergized to kill MM cells. Taken together, our data identified an “ EZH2 High t(4;14) +” signature in MM patients with a poor outcome who may benefit from combination therapy of MAK-683 with melphalan or epidrugs.

Label-Free High Dimensional Single Cell Morphological Profiling of Different Hematological Malignancies By Ghost Cytometry

Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern 'omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6) (Figure 1). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information (Figure 2). For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified unique cell populations across blood cell populations that were distinct from the healthy BM-MNC controls. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Keywords: flow cytometry, acute leukemia, multiple myeloma, label-free cell analysis, biomarker, liquid biopsy, ghost cytometry, artificial intelligence, machine learning Figure 1. Research design overview. Bone marrow samples from patients and non-diseased controls will be analyzed by Ghost Cytometry to assess disease by conventional markers and identify novel morphometric subpopulations. Morphological information is acquired as label-free GMI signals without any labeling. The label-free GMI information is transformed into two-dimensional space by UMAP dimensionality reduction method to identify subpopulations of interest. Both samples are stained with known cell surface markers for validation of unsupervised classification. Figure 2. Identification of disease-specific cell subpopulations by unsupervised approach. Using an unsupervised machine learning technique called UMAP, label-free morphologically distinct cellular subpopulations can be identified in B-ALL, AML, MM when compared to healthy donor controls (left panels, inside the dotted circles). For validation of the subpopulations, known cell surface markers (CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM) and cell trace marker (CellTracker) were overlaid. The intensity increases monotonically through the colormaps. The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50–75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

High-throughput analysis of glycan sorting into extracellular vesicles

Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.

Single-nucleotide polymorphism profiling by multimodal-targeted next-generation sequencing in methotrexate-resistant and -sensitive human osteosarcoma cell lines

Introduction: Methotrexate (MTX) is one of the most important drugs included in the first-line protocols to treat high-grade osteosarcoma (HGOS). Although several polymorphisms have been reported to be associated with drug response or MTX-related toxicity in pharmacogenetic studies, their role in the development of MTX resistance in HGOS is still unclear.Methods: Therefore, in this study, 22 single nucleotide polymorphisms (SNPs) in 4 genes of the folate metabolism, 7 MTX transporter genes, and 2 SNPs of the tumor protein p53 (TP53) gene were investigated using a custom multimodal-targeted next-generation sequencing (mmNGS) approach in 8 MTX-resistant and 12 MTX-sensitive human HGOS cell lines. The panel was validated by TaqMan genotyping assays.Results: High instability of TP53 rs1642785 was observed in all U-2OS/MTX variants. Allele changes of the solute carrier family 19 member 1/replication factor C subunit 1 (SLC19A1, previously known as RFC1) and rs1051266 were identified in all Saos-2/MTX-resistant variants in both DNA- and RNA- derived libraries compared to the parental Saos-2 cell line. Allele changes of methylenetetrahydrofolate reductase (MTHFR) rs1801133 were identified only in the RNA-derived libraries of the two U2OS variants with the highest MTX resistance level. Significantly upregulated gene expression associated with the development of MTX resistance was revealed for dihydrofolate reductase (DHFR) whereas SLC19A1 was downregulated. In addition, a fusion transcript of DHFR (ex4) and MutS Homolog 3 (MSH3) (ex9) was identified in the RNA libraries derived from the two U-2OS variants with the highest MTX resistance level.Conclusion: This innovative mmNGS approach enabled the simultaneous exploration of SNPs at DNA and RNA levels in human HGOS cell lines, providing evidence of the functional involvement of allele changes associated with the development of MTX resistance.

Targeting BCL-XL with a Novel VHL-Based BCL-XL Degrader DT-2216 in Pre-Clinical JAK2-Mutant AML Post-MPN Models

INTRODUCTION Outcomes of acute Myeloid Leukemia (AML) progressed from JAK2-mutant ( JAK2-mut) myeloproliferative neoplasms (MPNs) (post-MPN AML) are dismal, with limited responsiveness to cytotoxic therapies, hypomethylating agents, BCL-2 inhibitor or JAK2 inhibitor, with a median overall survival of less than 1 year ( Dunbar, et al. Blood, 2020). BCL-xL has been found to be overexpressed in cells from JAK2-mut MPN patients ( Petiti et al. J Cell Mol Med, 2020), thus making it a potential therapeutic target. However, the clinical utility of navitoclax, a BCL-xL and BCL-2 dual inhibitor, as demonstrated in combination with JAK2 inhibitor ruxolitinib in patients with myelofibrosis ( Harrison et al. J Clin Oncol, 2022), has been notable for an on-target thrombocytopenia due to the essential role of BCL-xL in platelet survival. In contrast, DT2216, a VHL-recruiting BCL-xL PROTAC, exhibits potent degradation of BCL-xL in leukemia cells while sparing platelets due to minimal expression of VHL in platelets ( Khan et.al Nat. Med, 2019). Consequently, DT2216 may represent an attractive strategy to target BCL-xL, reducing the apoptotic threshold and increasing responsiveness to chemotherapy selectively in tumor cells. In this study, we evaluated the pre-clinical efficacy of DT2216 in combination with ruxolitinib, 5-azacytidine (AZA), or MCL-1 inhibitor S63845 in JAK2-mut AML models. METHODS To validate the dependency of BCL2L1 (gene encoding BCL-xL) on JAK2-mut AML, we analyzed genome-wide CRISPR screen data, RNA-seq data from the DepMap portal and Beat AML 1.0 cohort. To assess the efficacy of DT2216-based treatments, including DT2216 alone, DT2216/AZA combination, DT2216/ruxolitinib combination, and DT2216/S63845 combination, we conducted the Cell Titer Glo (CTG) assay using JAK2-mut AML cell lines and primary leukemia samples. Since most JAK2-mut AML patients are resistant to ruxolitinib, we included ruxolitinib-resistant (ruxo-re) cell lines. The combination index (CI) values obtained at the ED 50, ED 75, and ED 90 were used to determine synergism (CI <1) between the agents, with lower values indicating stronger synergism. Clonogenic assays with three distinct JAK2-mut AML patient samples were performed using DT2216 and/or AZA to assess the anti-leukemia effects on hematopoietic stem and progenitor cells (HSPCs). Western blot was used to determine BCL-xL degradation and measure the expression of BCL-2 and MCL-1 after drug treatments. RESULTS The analysis of BCL2L1 gene expression (p<0.05, JAK2-mut AML cell lines versus other AML cell lines; p<0.01, post-MPN AML versus de novo AML) and BCL2L1 CRISPR gene effect (p<0.05, JAK2-mut AML cell lines versus other AML cell lines) confirmed the role of BCL-xL as a target in JAK2-mut AML. DT2216 as a single agent significantly reduced cell viability in JAK2-mut AML cell lines, with an average IC 50 of 1.61 μM in parental cell lines (range 0.74 - 2.35 μM) and an average IC 50 of 5.37 μM in ruxo-re cell lines (range 1.29 - 8.99 μM) at 72 hours. Notably, DT2216 also markedly reduced cell viability in primary samples (n=3), with an average IC 50 value of 1.21 μM (range 0.41 - 2.59 μM) at 24 hours. Moreover, the average CIs of DT2216/S68425 combination, DT2216/AZA and DT2216/ruxolitinib combination were 0.13 (range 0.07 - 0.17), 0.17 (range 0.61 - 0.22), 0.36 (range 0.14 - 0.54), respectively at 72 hours. Clonogenic assays in three primary JAK2-mut AML samples demonstrated a 69% (range 58.3 - 77.5%) reduction of colony count when treated with DT2216 alone (50 nM) (p<0.001, versus DMSO group) and 76% (range 70.8 - 79.8%) reduction when treated with the combination of DT2216 (50 nM) with AZA (100 nM) (p<0.0001, versus DMSO group) (Fig. 1A). Western blot analysis demonstrated efficient degradation of the BCL-xL protein, with average reductions of 78.5% (range: 70 - 90%), 82.75% (range: 68 - 96%) (Fig. 1B), and 86.75% (range: 83 - 97%) after 24 hours of treatment with DT2216 alone or in combination with AZA or ruxolitinib in both the parental and ruxo-re SET2 and HEL cell lines. CONCLUSIONS These findings highlight the promising efficacy of DT2216 in JAK2-mut AML, as evidenced by reduced cell viability, on-target degradation of BCL-xL, and synergistic anti-leukemia effects when combined with AZA, ruxolitinib or MCL-1 inhibitor. These results provide valuable insights into future therapeutic strategies for the treatment of JAK2-mut AML, particularly in the context of post-MPN AML.

Redundancy of Glycogen Synthase Kinase 3 in Lymphoma Cell Viability, Proliferation, and the Cytotoxicity of Elraglusib

Introduction Glycogen synthase kinase 3 (GSK3), a ubiquitously expressed serine/threonine kinase, is considered a therapeutic target in non-Hodgkin lymphomas (NHL) due to the poorer survival of patients with higher tumor expression, and the previously observed lethality of GSK3 ‘knock-out’ lymphoma cell lines. Elraglusib (formerly 9-ING-41), a small molecule reported to inhibit the GSK3β paralog, reduces proliferation and induces apoptosis in NHL cell lines, and is undergoing early-phase clinical trials (Wu et al, Blood 2019). However, in recent cell culture studies, the anti-lymphoma effects of elraglusib could not be phenocopied by structurally unrelated, small-molecule GSK3 inhibitors, raising questions about the importance of GSK3-dependent pathways in lymphoma biology and treatment (Coats et al, Cell Commun. Signal 2023). To further validate the lack of impact of GSK3 reduction on lymphoma viability and improve our understanding of the pharmacological properties of elraglusib, we have investigated the effects of reducing GSK3α, GSK3β, or both paralogs concomitantly in lymphoma cells, using paralog-specific and dual GSK3A/B genetic ablation techniques. Additionally, we performed cell-free kinase assays to explore the relative sensitivity of GSK3α and GSK3β as targets of elraglusib. Methods In the lymphoma cell line Karpas-299 (K299), single paralog GSK3A and GSK3B knockdowns (KD) were generated using commercial lentiviral shRNA transfection with subsequent puromycin-selection. Dual paralog KD (both GSK3A and GSK3B)were generated by transfecting stable GSK3A KD cells with a second commercial GSK3B lentiviral shRNA and subsequent neomycin-selection. KD efficiency was quantified by total-GSK3 immunoblot and apoptosis by poly (ADP-ribose) polymerase (PARP) cleavage immunoblot. Cell viability and proliferation were assessed by MTS assay following 72-hour incubations in complete media (RPMI 1640, 10% FBS, 100units/ml penicillin/100µg/ml streptomycin). Paralog-specific cell-free kinase assays were performed using 32P-γ-ATP, phospho-glycogen synthase-peptide-2 substrate and purified recombinant GSK3α and GSK3β. Results Viable and stable K299 single-paralog ( GSK3A or GSK3B) and dual-paralog ( GSK3A and GSK3B) KD cell lines were obtained. KD efficiency was quantified by immunoblot and compared to the parental cell line control for GSK3α (≈30% of control), GSK3β (≈20% of control) and dual GSK3αand GSK3β KD (≈35% control and ≈15% control respectively). All KD cell lines proliferated similarly to parental cell line cells by MTS assay (control vs GSK3B KD normalized to control, mean difference -5.1% (95% CI -11.7 to +1.37 p=0.14), control vs GSK3A/B KD +3.67% (95% CI -2.87 to +10.2 p=0.348)). Elraglusib 5µM induced apoptosis with a significant increase in cleaved/intact PARP (2.35x, p<0.0001) and reduced cell viability in K299 parental cell line and dual GSK3A/B KD cells (IC 50 0.158µM parental control vs 0.124µM dual GSK3A/B KD). The equivalence of paralog inhibition (GSK3β IC 50 0.494µM and GSK3α IC 50 0.446µM) was confirmed in cell-free 32P-γ-ATP kinase assays. Conclusion K299 cells do not depend upon high levels of GSK3 for survival or proliferation. This data extends previous findings regarding the redundancy of GSK3-dependent signalling in lymphoma biology and further suggests that limited therapeutic benefit may be derived from drugs specifically targeting the activity or expression of this protein kinase. The IC 50 of elraglusib remains similar despite a significant reduction in levels of GSK3, again suggesting that GSK3 is not its cytotoxic target. Importantly, elraglusib retains therapeutic potential in lymphoma however GSK3 is not a surrogate marker for its efficacy and additional studies are required to elucidate the mechanisms through which it mediates its cytotoxic effects.

CDK9 Inhibition Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma (MCL)

Introduction: Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib [ibr]) have shown clinical efficacy in MCL. However, resistance to BTK inhibitors is inevitable. Cyclin-dependent kinases (CDKs)are Ser/Thr kinases that associate with specific cyclins during the cell cycle to regulate its progression. We and others have shown that selective targeting of CDK9, a pTEFb component which facilitates RNA transcription, downmodulates MYC and MCL1 in malignant B-cells. Here, we investigated the effects of a selective CDK9 inhibitor, AZD4573, in ibr-naive and ibr-resistant MCL in vitro and in vivo. Methods: MCL cell lines (Mino, JeKo-1) were cultured in increasing doses of ibr for ~6 months to generate resistance. MCL cell lines were treated with AZD4573 and assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), and protein and RNA expression. Primary MCL cells were incubated for 24 h in CD40L/BAFF-conditioned media prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected IV in NSG mice and tracked by flow cytometry (CD5 + CD19 + CD45 +). Upon MCL detection in the peripheral blood (~1-2%), mice began treatment with AZD4573 (25 mg/kg IP once or twice weekly) or vehicle control. Splenocytes were harvested and RNA was isolated and analyzed by RNA-Seq/RT-PCR. Additionally, primary circulating MCL cells were collected from a patient treated with one dose of AZD4573 (at baseline; 4 and 7 h post-infusion and day 7) and analyzed by scRNA-Seq. Results: Treatment with AZD4573 potently inhibited proliferation and induced apoptosis of both ibr-sensitive and resistant MCL cell lines in vitro (IC 50 ~ 2-15 nM). AZD4573 also induced apoptosis of primary MCL cells in stromal conditions. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis as measured by cleaved PARP. AZD4573 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect. CDK9 inhibition resulted in rapid and dose-dependent downregulation of MYC and MCL1 mRNA and protein levels in MCL cell lines and primary cells. Treatment with AZD4573 but not with ibr extended survival of ibr-resistant PDX mice without significant toxic events. RNA-Seq analysis of the MCL splenocytes harvested from these mice after 3 weeks of AZD4573 treatment demonstrated that gene sets related to transcription-factor activity were most enriched in the treated samples, consistent with known drug mechanism. TNFa/NFκB, TGF-β and PI3K/AKT/mTOR signaling pathways were most downregulated. We have shown that CDK9 inhibition leads to epigenetic reprogramming in neoplastic B-cells, resulting in upregulation of a subset of superenhancer-associated oncogenes, i.e. MYC, PIM kinaseand others (Thieme et al, 2023). Consistent with this notion, splenocytes derived from AZD4573-treated mice exhibited upregulation of MYC-dependent targets and OxPhos at the time of sacrifice. A number of genes relating to components of the electron transport chain were upregulated ( ATP5F1D, NDUFS7, ATP5MC1 as the top three). This suggested that these pathways may govern resistance to CDK9 inhibition in MCL over time. To further explore this, parental and ibr-resistant MCL cell lines were treated with low doses of AZD4573 (~2 nM). After 48 h, CDK9 inhibition was associated with increased oxygen consumption rates (OCR) in Seahorse analysis. This was not accompanied by increased cell proliferation by cell cycle analysis or Ki-67 staining. IACS-010759, an inhibitor of complex I of the ETC which targets OxPhos, demonstrated synergy with AZD4573 in parental and ibr-resistant MCL cell lines. Single-cell RNA-Seq analysis of primary MCL cells obtained from a patient treated with AZD4573 on a clinical trial revealed rapid downmodulation of NFκB signaling (at 4 h) and MYC transcriptional targets and PI3K/AKT/mTOR signaling pathways (at 7 h). Interestingly, we observed partial recovery of MYC, mTOR pathways and induction of OxPhos by day 7 (prior to next planned dose), validating our pre-clinical findings. Conclusion: CDK9 inhibition with AZD4573 induced apoptosis, downregulated MYC and MCL1 and NFκB signaling, and overcame ibr resistance in preclinical MCL models. This was also noted in a sample obtained from a clinical trial. Prolonged CDK9 inhibition led to metabolic reprogramming towards OxPhos, which thus can serve as a therapeutic target in MCL.

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

A Necroptosis Inhibitor Functions As a Ferroptosis Inducer in Drug-Resistant Myeloid Malignancies

Introduction: Acute myeloid leukemia (AML) is associated with poor prognosis, especially in elderly patients unfit for intensive chemotherapy. AML chemotherapy agents, including venetoclax, a BH3-mimetic, are designed to induce cell death through apoptosis. Unfortunately, AML cells eventually develop a resistance to BCL-2-dependent apoptosis which causes clinical disease recurrence. Alternative cell death pathways like ferroptosis and necroptosis are lytic forms of cell death mediated by plasma membrane lipid oxidation or MLKL phosphorylation (p-MLKL), respectively, both of which disrupt plasma membrane integrity. Because apoptosis and ferroptosis involve different molecular pathways, ferroptosis-inducing therapies provide an opportunity to overcome therapy resistance in AML patients. Here, we show how necroptosis inhibitor targeting basal necroptosis signal selectively induces ferroptosis in venetoclax-resistant AML cells causing cell death. Methods: We established venetoclax-resistant myeloid cell lines by exposing cell lines to increasing doses of venetoclax. We evaluated resistance to venetoclax-induced apoptosis by measuring IC50 after Annexin-V/PI staining. We screened venetoclax-resistant myeloid cell lines and primary AML cells (n=77) for basal activation of necroptosis by pMLKL or phosphorylated RIP3 (pRIP3) western blotting. Xenotransplantation was performed to evaluate the bone marrow engraftment potentials of basal activated necroptosis primary AMLs. Necrosome inhibitor (Zharp-99) was tested to estimate IC50 for cell death induction in myeloid cell lines after Annexin-V/PI staining. Ferroptosis inhibitors (ferrostatin-1 and lipoxstatin-1) and apoptosis inhibitor (zVAD-FMK) were used to determine which death pathway was induced by necroptosis inhibitors in AML. Results: We established venetoclax-resistant myeloid cell lines (THP1-VR and HL60-VR), which showed a significant shift in IC50 for venetoclax-induced apoptosis compared to parent cell lines. Venetoclax-resistant cell lines acquired an upregulation of known anti-apoptotic proteins; BCL-2, BCL-XL, and MCL-1. Venetoclax-resistant cell lines upregulated pRIP3 signal, a known necroptosis driver, by western blot. Necrosome screening of primary AML samples revealed 73 AMLs (94%) showed basal necroptosis signal confirmed by pMLKL with heterogeneous intensities (Figure. A). Xenotransplantation confirmed engraftment of basal necroptosis-activated primary AMLs is achievable and with retention of necroptosis signal at engraftment. The basal necroptosis signal in venetoclax-resistant cell lines and primary AML samples drove us to evaluate necroptosis inhibitor Zharp-99 (RIP3K inhibitor) IC50 for venetoclax-resistant cell lines. Compared to parent cell lines, venetoclax-resistant cell lines became susceptible to cell death by necrosome inhibition. Surprisingly, the cell death induced by Zharp-99 was not from apoptosis but through ferroptosis, confirmed by the venetoclax-resistant myeloid cells being rescued from cell death by ferroptosis inhibitor (ferrostatin-1 and lipoxstatin-1) (Figure B). Conclusion: Basal activation of necrosome is observed in venetoclax-resistant myeloid cell lines and primary AML patient samples. We found necroptosis inhibitor Zharp-99 promoted cell death in necrosome-activated venetoclax-resistant cell lines. Our data suggest necroptosis inhibition targeting RIP3K activity has a novel function of inducing ferroptosis in myeloid malignancies resistant to apoptosis. RIP3K inhibitor, as a novel function of ferroptosis inducer in apoptosis-resistant myeloid cell lines, opens a new therapy opportunity to treat apoptosis resistance in AML patients. We plan to investigate in vivo effects of necrosome inhibitors using our xenotransplant model.