L428 Cells Research Articles

Preclinical In Vivo Imaging of Multiple Myeloma with [ 18f]Fluorohomoleucine (FHL) PET/CT

Purpose: Imaging of patients with multiple myeloma (MM) remains challenging due to its multi-focal nature affecting bone marrow, bone structures and soft tissue. As a result, current diagnostic methods like X-rays, CT and MRI scans only provide a partial understanding of disease activity and treatment response, lacking a comprehensive overview. Although 2-deoxy-2-[ 18F]fluoro-D-glucose (FDG) positron emission tomography (PET) has become the standard for detecting multifocal cancer spread, its use in MM patients is limited due to metabolic heterogeneity of MM and limited sensitivity in detecting extramedullary disease leading to false negative and false positive scans. To overcome these limitations, we developed a novel radiopharmaceutical L-[ 18F]Fluorohomoleucine (FHL) targeting LAT1 amino acid transporter. LAT1 has been significantly correlated with the proliferative activity of MM cells, poor prognosis and response to treatment in MM patients, presenting an appealing molecular target for assessing disease activity. Methods: FHL was synthesized as described by Nodwell et al. (J Nucl Med, 2019) and FDG was obtained from BC Cancer. LAT1 expression in multiple myeloma (MM) cell lines was confirmed by flow cytometry. Cell uptake assays were conducted using luciferase-transfected MM1S, L363, NCI H929 and AMO1 MM cells, and 2-amino-bicyclo[2.2.1]heptanes-2-carboxylic acid (BCH) was used as a blocking agent. For in vivo studies, male NRG mice were injected either with 1 x 10 6 MM cells intravenously (i.v.) via the caudal vein or subcutaneously (SQ) with 1 x 10 7 cells. Tumor engraftment was monitored weekly on an in vivo bioluminescence imager Perkin Elmer's IVIS Ilumina 5, starting at 2 weeks post-injection (p.i.). Biodistribution and PET/CT imaging studies were conducted at 4-6 weeks p.i. Results: All MM cells tested exhibited LAT1-specific FHL uptake in vitro with L363 and NCI H929 demonstrating the highest tracer uptake and average blocking efficacy over 83% and 93%, respectively. We were able to observe weekly progression of disease in the i.v. cohort using IVIS imaging. Biodistribution and PET imaging studies revealed FHL accumulation in the SQ xenografts, pancreas and excretion via the urinary tract. The SQ tumor uptake values were 4.53±0.82 %ID/g in AMO1 xenografts, 5.55±0.82 %ID/g in NCI H929, and 6.37±0.70 %ID/g in L363 tumors. In the i.v. cohort, the mouse intravenously injected with L363 cells demonstrated accumulation of radioactivity in the skull and vertebrae, as anticipated from the IVIS images. Furthermore, preliminary data suggests that FHL was able to detect a more extensive bone and lymphatic involvement than FDG in MM xenografts. Conclusion: We have demonstrated the ability of FHL for in vivo imaging of three human multiple myeloma tumors in SQ murine models, with the tumor uptake ranging from 4.53±0.82 %ID/g in AMO1 xenografts to 6.37±0.70 %ID/g in L363 tumors. Work is underway to establish the utility of this tracer in detecting disease dissemination in the orthotopic murine models of multiple myeloma, with promising preliminary results presented herein.

Macropinocytosis As a Means of Selectively Targeting RAS-Mutant Multiple Myeloma

Introduction Roughly, 50% of multiple myeloma (MM) is driven by mutant RAS, both at presentation and more at relapse. Currently, in MM there are no clinical approaches to target this disease subset and ongoing strategies to inhibit RAS function or downstream effectors have had limited clinical success in other tumors. This has prompted our team to look into other more indirect modes of RAS-selective targeting. Oncogenic RAS-driven cancers use a unique actin-driven endocytosis pathway, termed macropinocytosis (MP), to increase the nutrient supply and sustain proliferation. To create a strategy for targeting RAS mutant MM, we have investigated whether RAS-mutant MM cells undergo MP and whether this process can be exploited as a therapeutic vulnerability, by utilizing this pro-oncogenic biochemical mechanism to selectively deliver a toxin to the MM clone using a MP selective monobody-drug conjugate (MDC). Methods The MDC relies on a proprietary fibronectin-based scaffold (monobody) identified based on its physiochemical properties that facilitates MP selective internalization, and therefore targets RAS-mutant MM tumor cells, with a very favorable pharmacokinetic profile. MMAE was attached to the monobody with a cathepsin-cleavable linker and a maleimide group for conjugation to the C-terminal free cysteine on the monobody protein (MDC-MMAE). MDC-MMAE was pre-clinically evaluated both in-vitro and in-vivo, in human MM cell lines, patient cells and a doxycycline inducible mutant KRAS cell system. Results In-vitro cell line and patient cell studies demonstrated that RAS-mutant MM cells undergo MP whereas wild type (WT) cells do not. To understand the role of mutant RAS in driving uptake of the MDC, a doxycycline-inducible mutant KRAS V12 KMS11 system was established. The functionality of the inducibility of the cell system was verified using protein expression and membrane ruffling in the presence of doxycycline. Fluorescently-labeled monobody (MDC-Cy5.5) and TMR-Dextran underwent a high level of uptake in the presence of induced mutant RAS. In contrast, in conditions where MP was absent, no uptake of MDC-Cy5.5 and TMR-Dextran was seen. Furthermore, when KMS11 KRASV12 and WT KMS11 were treated with MDC-MMAE, a 2-fold increase in cell death was observed in the mutant RAS cell line compared to wild type RAS. In-vivo studies showed that the pharmacological properties of the drug when conjugated to the monobody enabled faster serum clearance compared to antibody drug conjugates (ADCs) and nanomaterials, while maintaining cellular impermeability allowing enhanced drug uptake via MP. Importantly, in-vivo the MDC-MMAE technology had an improved toxicity profile compared to ADC-MMAE, increasing tolerable drug delivery by upwards of 15-fold. To test the anti-tumor effects in-vivo, the L363 (NRAS Q61), WT KMS11, and KMS11 KRASV12 cell lines were subcutaneously injected into MM murine models. After tumors were established, 0.5mg/kg MDC-MMAE was administered every 7 days. Mutant RAS L363 and KMS11 KRASV12 tumors treated with MDC-MMAE exhibited roughly a 6-fold decrease in tumor size after 21 days while WT KMS11 tumors showed no significant difference in tumor volume. MDC-MMAE at 5mg/kg showed complete tumor regression as a single agent in L363 tumors. Improved outcomes were seen with MDC-MMAE in combination with standard of care options at lower doses. Additionally, MDC-MMAE has displayed enhanced immunogenic cell death that has not been seen with other chemotherapeutic options, making it an ideal partner for immunotherapy combination. Conclusion Limited clinical options exist for the treatment of mutant RAS MM tumors. We show that by taking advantage of MP, a process upregulated in RAS-mutant cancers, MDC-MMAE specifically targets and inhibits MM growth and improves survival in murine models. It can also be combined with other MM standard of care drugs and with immunotherapy drugs to have a potentially important clinical effect. In collaboration with Tezcat Biosciences, the MDC-MMAE technology has achieved a phase II SBIR for advancement to clinical application.

Multispecific Hybrid T Cell Receptors for Sensitive Targeting of Cancer

CAR T cells represent a promising therapy for a growing list of tumors. Despite high initial response rates, 50-60% of patients relapse within 1 year after CAR T cell infusion. Heterogeneity of target antigen expression can lead to outgrowth of tumor cells with low or negative antigen levels. Conventional CARs require high antigen levels for efficient T cell activation and tumor elimination. By contrast, T cell receptors (TCRs) are 10-100 times more sensitive than CARs. To achieve greater antigen sensitivity, we and others designed synthetic hybrid T cell receptors by fusing antibody-based recognition domains to TCRa and TCRb constant chains. Here we extend the initial monospecific design to incorporate two antigen recognition domains. We evaluated 2 monospecific hybrid receptors, one with VH and VL domains fused to TCRa and TCRb constant chains respectively (Split TCR/CAR) and a second where the entire scFv (VL-linker-VH) is fused only to the TCRa chain (Full TCR/CAR) leaving TCRb available for a second scFv. We used based editing to knock out endogenous TCRa and b chains to avoid mispairing. Using CD19 as a model target we found that both TCR/CAR designs lacked tonic signaling, induced TCR-like changes in Ca+ flux, more efficiently phosphorylated LAT, and exhibited superior recognition of Nalm-6 clones expressing a range of CD19 levels compared to conventional CD19 28z or 4-1BBz CARs. Interestingly, the Full TCR/CAR outperformed the Split format in these assays. Using soluble bilayer and TIRF microscopy, we demonstrated that unlike CARs, TCR/CAR T cells recapitulate a classical TCR-like immunological synapse. I n vivo studies using NSG mice engrafted with CD19+ Raji cells demonstrated potent anti-tumor efficacy, and the Full format outperformed the Split TCR/CAR. Capitalizing on the superiority of the Full TCR/CAR format, we next designed bispecific hybrid receptors composed of two scFvs of different specificities fused to the TCRa and TCRb chains. Multiple Myeloma is particularly suitable for bispecific targeting as relapse following CAR T cell therapy is common and multiple target antigens have been described. We designed bispecific Full TCR/CARs specific for pairs of myeloma antigens (BCMA/SLAMF7 and BCMA/GPRC5D) and expressed them in primary T cells. We used base editing to knock out SLAMF-7 which can be expressed in T cells and could cause fratricide. Bispecific TCR/CAR receptors conferred sensitive T cell recognition of target cells expressing BCMA and SLAMF-7 as measured by cytokine production, proliferation, and cytotoxicity. T cells equipped with these novel bispecific receptors maintained function when exposed to L363 cells BCMA ko or SLAMF-7 ko demonstrating that both antigen binding domains can trigger T cell functions against target cells. In vivo studies using NSG mice implanted with a mixture composed of 33% of Nalm-6 BCMA+, Nalm-6 SLAMF-7+ and Nalm-6 BCMA+SLAMF-7+ cells to mimic target antigen expression heterogeneity and antigen loss showed superior anti-tumor efficacy for bispecific TCR/CAR cells compared to conventional BCMA and SLAMF-7 BBz CARs. Our hybrid bispecific TCR/CAR also demonstrated superior anti-tumor efficacy compared to a previously described, and systematically optimized bispecific CAR format (Zah et al., 2020). Bispecific hybrid T cell receptors embedded with natural TCR signaling machinery represent a promising therapeutic option to address antigen downmodulation/loss and antigen heterogeneity for treatment of cancer.

Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host’s immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.

Discovery of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Ka/HDAC6 for the treatment of cancer

Recently, PI3K and HDAC have been considered as promising targets for the cancer therapy. A couple of pan-PI3K/HDAC dual inhibitors have been developed as a new class of anticancer agents. Herein, we discovered a new series of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Kα/HDAC6. All the derivatives exerted dual-target inhibitory activities. Particularly, in the enzymatic selectivity assay, compound 21j was identified as a subtype-selective PI3Kα/HDAC6 dual inhibitor (IC50 = 2.9 and 26 nM against PI3Kα and HDAC6, respectively), which displayed high potency against L-363 cell line with IC50 value of 0.17 μM. In addition, 21j significantly inhibited phosphorylation of pAkt(Ser473) and induced accumulation of acetylated α-tubulin while having a negligible effect on the levels of acetylated Histone H3 and H4 at nanomolar level. Attributed to its favorable in vitro performance, 21j has the potential to alleviate the adverse effects resulted from pan-PI3K inhibition and pan-HDAC inhibition. It is valuable for further functional investigation as an anti-cancer agent.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease.

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest.

Critical Role of Hodgkin Lymphoma Cells-Derived Large and Small Extracellular Vesicles in the Regulation of the Immune-Inflammatory Microenvironment

Introduction: Classical Hodgkin Lymphoma (cHL) is characterized by a low number of CD30+ malignant cells and an extensive immune-inflammatory microenvironment. However, this unique histopathological picture and its pathogenesis are still partially understood. Extracellular vesicles (EVs) are submicron particles delimited by a lipid bilayer that promote (tumor) intercellular communication and carry bioactive molecules that can influence the immune system. Based on size and biogenesis mechanism, Small (S-) and Large (L-) EVs (30-200 nm and 0.2-10 µm, respectively) have been identified. Extracellular Adenosine Triphosphate (eATP), a key component of the tumor microenvironment, plays a role in biogenesis and dynamics of EVs. Here we aim to elucidate the role of cHL cell-derived EVs in the education of the immune microenvironment. Methods: cHL L-428 cell line was cultured in exosome-free medium for 48 hours at 37°C in the presence or absence of eATP (1 mM). L-428 cell blebbing has been detected by fluorescence microscopy (DAPI and FITC-Cholera Toxin B subunit (CTxB) labeling). L-EVs and S-EVs were isolated from the cell culture supernatant by differential ultracentrifugation (2.800g for 2.8K and 20.000g for 20K L-EVs, and 100.000g for 100K S-EVs) and purified by iodixanol density gradient. L- and S-EVs were then characterized by Tunable Resistive Pulse Sensing (TRPS), single EV-flow cytometry, and MACSPlex (bead-based antigen binding array). Functionally, normal peripheral blood mononuclear cells were in vitro cultured with titrated amount of isolated L- and S-EVs (from 1:5 to 1:20) for 24 hours. The intracytoplasmic interleukin-1β and interferon-γ production by CD14+ monocytes and CD3+/CD4+/CD8+ T cells was assessed by flow cytometry. Results: Our results showed that human cell line L-428 is able to produce L-EVs through membrane blebbing (Figure 1A). According to EV size distributions, both 2.8K and 20K L-EVs displayed a size range from 1 to 4 µm whereas S-EVs were enriched in particles of 150-200 nm diameter. Interestingly, the amounts of S-EVs shed by L-428 cells were significantly higher compared to the number of L-EVs. Considering the EV surface protein phenotype, S-EVs express all the EV-related tetraspanin proteins (CD9, CD63, and CD81). By contrast, L-EV express CD63 and CD81 only. Additionally, in L-EVs we observed an enrichment of cancer-related markers including SSEA-4, CD44, CD24, CD29 and CD133-1 in comparison with the S-EV counterpart. Of note, the expression of selected immune-related molecules such as CD86 and CD40 was higher in the 2.8K and 20K L-EVs as compared with that of the 100K S-EVs. The HLA-II molecules were expressed by all fractions, with the highest expression in 2.8K L-EVs. Along with PD-L1, the CD30 HL-associated marker was expressed by all EV fractions at the same extent. When we tested the effects of eATP on EVs produced by L-428 cells, we found a highly decline in the expression of EV-surface proteins from all EV sub-fractions, being most noticeable in L-EVs. Importantly, only PD-L1 expression was increased in the 20K L-EVs after eATP treatment. Functionally, at variance with S-EVs (Figure 1B), L-EVs highly stimulate the intracytoplasmic production of interleukin-1β by monocytes. Conversely, L-EVs derived from e-ATP treated L-428 cells fail to stimulate the intracytoplasmic production of interleukin-1β by CD14+ monocytes, which instead was promoted by the e-ATP treated S-EV counterparts. Of note, neither L-EVs nor S-EVs stimulated interferon-γ production by CD14+ monocytes. No intracytoplasmic production of the selected inflammatory cytokines has been observed when T-cells treated with EVs were analyzed. Conclusions: These results demonstrate that L-EVs from HL cells display a cancer- and immune-related phenotype and play a role in the modulation of the immune microenvironment by promoting the production of inflammatory cytokines by monocytes. This might be partially due to the high expression of CD44 on the surface of L-EVs, since it has been previously demonstrated that cancer-derived soluble CD44 may trigger monocyte/macrophage interleukin-1β production. Interestingly, eATP plays a role in shaping the immune modulatory ability of L- and S-EVs. These findings shed light on the role of HL-derived EVs in the establishment of the immune-inflammatory microenvironment and provide novel insight into clinically relevant EV driven mechanisms in HL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Modulation of the CGAS-Sting Anti-Tumor Immune Response Pathway in an NF-KB-Dependent or -Independent Manner As a Potential Therapeutic Target in Classical Hodgkin Lymphoma (cHL)

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remains unknown to date. In this study, we hypothesized that NF-kB pathway, a critical player in cHL oncogenesis, may regulate anti-tumor immune responses through modulation of the cGAS-STING pathway activity and gene expression of the relevant interferons and chemokines. Methods: STING expression was immunohistochemically analyzed in a pilot study group of 32 untreated patients with cHL and available tissue from diagnostic lymph node biopsies using a previously validated monoclonal antibody and standard methods. In addition, control lymphoid tissue (2 reactive lymph nodes, 2 tonsils) were used. At least 500 neoplastic Hodgkin & Reed-Sternberg (HRS) cells were counted and an arbitrary 10% cutoff was used for positivity. The in vitro system included 6 cHL cell lines (MDA-V, L-1236, L-428, L-540, HDLM-2, KM-H2). Gene expression (mRNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot analysis, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING and NF-kB genes was performed in all cHL cell lines using transient transfection (Nucleofector system, Amaxa, Lonza) with specific STING and NF-kB (RELA) siRNA constructs. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH), with RT-qPCR. Results: In reactive lymphoid tissue, STING expression was restricted to dendritic cells and a subset of T-lymphocytes with a cytoplasmic staining pattern, which served as internal positive controls in all immunostainings. Using an arbitrary 10% cutoff, STING was positive in the neoplastic HRS cells of cHL in 20 of 32 (63%) patients with a predominant cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L-1236, L-428 and HDLM-2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Silencing of NF-kB (RELA) resulted in activation of cGAS-STING pathway as shown by IRF3 activation (phosphorylation) and increased IRF3 protein levels in L-428 cells, which was associated with increased CXCL10 mRNA levels. Treatment with Amlexanox resulted in downregulation of IFN-β and/or CXCL10 gene expression in cHL cell lines. Conclusion: The cGAS-STING pathway is activated and, at least in part, regulated by the oncogenic NF-kB pathway. STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analyzed for the expression and prognostic significance of STING and the final results will be available at the time of the meeting.

The Cancer-Related Circrna Cirs-7 (CDR1-AS) Is Differentially Expressed in CD138+ Cells of Patients with Plasma Cell Disorders and Predicts Unfavorable Overall Survival in Multiple Myeloma

Introduction: During the last decades, different non-coding RNAs have been implicated in the biology and prognosis of multiple myeloma (MM). Circular RNAs (circRNAs) constitute a class of non-coding RNAs with a high potential as molecular biomarkers. Recently, ciRS-7 (also known as CDR1-AS), an important circRNA regulating miR-7-5p and other cancer-related miRNAs, was shown to be downregulated in MM patients’ cells exhibiting acquired resistance to immunomodulatory drugs (IMiDs). Our goal was to study the expression of this circRNA in MM, compare the results with those of patients with smoldering myeloma (SMM) and monoclonal gammopathy of undetermined significance (MGUS) and evaluate its prognostic significance. Methods: Bone marrow aspirate (BMA) samples were collected from 177 adult patients with plasma cell disorders. The presence of high-risk cytogenetic abnormalities, namely del(17p), t(4;14), t(14;16), and (+1q), was assessed using fluorescence in situ hybridization (FISH). MM patients were classified according to the ISS and revised ISS (R-ISS) staging systems. Whole-body low-dose computed tomography was used to assess osteolysis. Next, CD138-positive selection of plasma cells among mononuclear cells of BMA samples was performed. Moreover, three human MM cell lines (L-363, H929 and U266) were propagated. Total RNA was extracted from CD138+ enriched BMA samples and the MM cell lines, and reverse-transcribed. A first-round PCR was used to pre-amplify ciRS-7 and GAPDH (i.e. reference gene) transcripts, before their quantification via real-time qPCR. In both reactions for ciRS-7 amplification, divergent PCR primers were used, to achieve high specificity and accuracy. ciRS-7 expression was measured in relative quantification units (RQU). Results: The cohort of patients consisted of 111 newly diagnosed MM, 35 SMM and 31 with MGUS. In total, 107 out of the 177 cases (60.5%) were male and 70 (39.5%) were female. The median age of all patients at diagnosis was 70 years (range: 35-93 years). According to the R-ISS staging system, 24 (21.6%) MM patients had R-ISS stage I, 62 (55.9%) R-ISS stage II and 25 (22.5%) had R-ISS stage III. Seventy-seven (69.4%) MM patients presented with osteolytic disease at diagnosis. Regarding front-line therapy of MM patients, 12/111 (10.8%) received proteasome inhibitor (PI)-based therapy, 7 (6.3%) received IMiD-based treatment, 74 (66.7%) were treated with combinations of PI and IMiD, and 18 (16.2%) received anti-CD38-based treatment. The intracellular levels of the ciRS-7 circRNA were different between MM, SMM and MGUS patients’ CD138+ plasma cells (median values were: for MM patients 0.40 RQU vs. 1.74 RQU for SMM patients vs. 2.79 RQU for MGUS patients; P<0.001). Moreover, ciRS-7 levels in U266 and H929 cells were much lower (2,165- and 26,616-fold) than in L-363 cells. ROC analysis showed that ciRS-7 expression can effectively discriminate between MM and SMM patients (AUC=0.65, 95% CI=0.56-0.75, P=0.007). ciRS-7 levels did not significantly differ among MM patients with different ISS or R-ISS stages and were not associated with any assessed cytogenetic abnormality or the presence of osteolytic lesions. Regarding the progression-free (PFS) and overall survival (OS), out of 106 MM patients with survival data, patients with high levels of ciRS-7 have lower survival probabilities. As shown in Figure 1, the median PFS of MM patients with high ciRS-7 (high: defined as higher of the median value) was 19 months vs. 25 months for patients with low ciRS-7 (lower values than the median) (P=0.046); similarly, the mean OS of ciRS-7 (high) MM patients was 25 months vs. 30.5 months for ciRS-7 (low) MM patients (P=0.008). Bootstrap multivariate Cox regression analysis showed that the unfavorable prognostic value of ciRS-7 expression with regard to OS is independent of R-ISS stage and age of MM patients (HR=2.6, BCa 95% CI=0.91-4.75, bootstrap P=0.041). Conclusions: Expression levels of ciRS-7, a cancer-related circRNA, in CD138+ plasma cells are significantly different among patients with MM, SMM and MGUS. Most likely, high ciRS-7 levels predict an unfavorable prognosis in MM, independently of the R-ISS stage and age. Combining our results with the limited existing literature providing evidence for ciRS-7 implication in response to IMiD therapy, its biological role in MM deserves further elucidation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MEN1703-Mediated PIM Kinases Inhibition Impairs Protumoral and Immunosuppressive Phenotype and Functions of Macrophages in Classical Hodgkin Lymphoma

Increased abundance of tumor-associated macrophages (TAMs) predicts shortened survival of patients with classical Hodgkin lymphoma (cHL). TAMs in cHL might support growth of the malignant, Reed-Sternberg (RS) cells and due to expression of PD-L1, contribute to T-cell exhaustion. In our previous studies, we identified almost universal expression of PIM-1/2/3 kinases (PIMs) in RS cells, but also noted that they are expressed in TAMs. In RS cells, PIMs support malignant cells survival and immune privilege. However, their role in the biology of cHL-TAMs remains to be explored. Herein, using an in vitro model of cHL-TAMs (RS-conditioned macrophages, RS-M) we characterize consequences of PIM inhibition for phenotype and functions of TAMs in cHL. THP1 cells and donor-derived monocytes were differentiated intomacrophages(Mϕ-0) using PMA and CSF-1 respectively. Mϕ-0 were next cocultured with L428 or L1236 RS cells under conditions prohibiting direct contacts. Compared to Mϕ-0, RS-M exhibited elevated expression of PIMs and genes involved in chemotaxis/immunomodulation (CCLs: 2, 5, 7, 8, 13, 17, 18 and 24), extracellular matrix organization (CD206, TGM2 and MMP-1, -7, -9 and -12), T cell exhaustion (PD-L1) and angiogenesis (VEGFA, PDGFB, CHI3L1/2). PIMs expression was also detected in primary cHL-TAMs. To assess role of PIM kinases in TAMs, RS-M were treated with a dual pan-PIM/FLT3 inhibitor, MEN1703, and subjected to transcriptional, biochemical and immunophenotype analyses. MEN1703 skewed gene expression profiles of RS-M toward pro-inflammatory (M1) macrophages and downregulated expression of genes associated with pro-tumoral (M2) macrophages. Consistently, PIM inhibition in RS-M downregulated expression or decreased activity of M2-associated molecules (CREB1, AKT, STAT3/6, CD163, CD206, CD209), and decreased PD-L1 levels by 32%. PIM inhibition hampered important functions of RS-M, including their ability to promote angiogenesis, matrix remodelling (collagen uptake), and eosinophil recruitment. In direct cocultures, RS-M decreased activity of T lymphocytes. This suppressive effect was alleviated in cocultures with the PIM inhibitor-treated RS-M. Our data suggest that PIMs support pro-tumoral and immunosuppressive phenotype of cHL-TAMs. Since PIM activity is required for RS cell survival and immune escape, these kinases are rational targets for therapy in cHL, suggesting the use of PIM inhibitors, such as MEN1703, as a possible therapeutic approach .

S101: GENETIC AND EPIGENETIC FACTORS DRIVING PRIMARY MEDIASTINAL B-CELL LYMPHOMA PATHOGENESIS AND OUTCOME

Background: Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive lymphoma affecting predominantly young female patients. Previous studies in this rare entity have focused on single genes or were limited in cohort size. Aims: To unravel the underlying genetic pathogenesis and its impact on outcome, we embarked on a comprehensive large-scale genetic investigation. Methods: Specimens of 486 previously untreated PMBCL patients were analyzed by paired tumor/normal whole-genome (WGS, n=14), whole-exome (WES, n=78) and targeted sequencing (TS, n=486). To understand the consequences of highly recurrent mutations in the chromatin-modifying gene ZNF217, we conducted functional and multi-omics analyses in CRISPR/Cas9 engineered cell lines. Results: WGS/WES revealed a complex genomic landscape in PMBCL with a median of 85 structural variants, a mutational burden of 5 mutations/Mb, 12 mutated coding candidate driver genes (CDG) and 4 focal somatic copy-number aberrations per sample (Fig.1a). Besides known targets, significant breakpoints were identified in genes previously not implicated in B-lymphomagenesis such as TOX and TP73 (36% and 21%). In addition, non-coding mutations clustered within the PAX5 enhancer region. With the identification of 50 recurrently mutated CDGs, we significantly expand the repertoire of known PMBCL drivers. The 10 most frequently mutated CDGs were SOCS1 (86%), B2M (67%), ITPKB (64%), ACTB (58%), STAT6 (58%), IGLL5 (56%), TNFAIP3 (53%), NFKBIE (49%), GNA13 (47%), and ZNF217 (36%), respectively. The operative mutational processes were attributed to aging, AID/APOBEC activity, defective MMR, and an unexpected infidelity of DNA-Polymerase ε. Next, we performed TS in 486 samples using a PMBCL-specific 106-gene panel. Recurrent lesions in 25 epigenetic modifiers were found in >90%, with ZNF217 being among the most frequently mutated genes (Fig.1b). After knockdown of ZNF217 in Karpas1106P and L428 cells, we demonstrated altered proliferation, migration, and apoptosis. Using mass spectrometry, we showed that ZNF217 is acting in a LSD1, CoREST and HDAC containing histone modifier complex. Accordingly, knockout of ZNF217 led to global changes in chromatin accessibility with an enrichment of differentially accessible motifs for crucial lymphoma-associated transcription factors, especially of the NF-κB, BATF/AP1, and IRF family, but also of CTCF, a major regulator of global 3D chromatin architecture. Resulting gene expression was characterized by changes in interferon-responsive genes and inflammation-associated transcription (Fig 1c). Clinical data were available for 329 cases, including 84 cases from clinical trials. Multivariate analysis using an IPI-corrected Cox regression model was performed. The estimated 5-year PFS and OS were 77% and 86%. Among the genetic lesions with the strongest association for poor outcome, we identified patients with mutatedCD58 having a significantly shorter survival (PFS: HR 2.96; p<.001; OS: HR 2.55; p=.006). In contrast, mutated DUSP2 indicated longer survival (PFS: HR 0.28; p=.002; OS: HR 0.15; p=.011) (Fig1d). Notably, DUSP2 mutated patients (25%) showed a similar outcome for CR rate, PFS and OS when comparing CHOP-like and intensified treatment regimens, suggesting no further benefit from treatment intensification in this very-low risk patient population. Image:Summary/Conclusion: Here, we present the genetic landscape of PMBCL highlighting a previously underappreciated role of chromatin modifying genes, identify novel treatment targets and provide a solid basis for guiding precision medicine approaches.

P863: GENERATION OF A FIRST-IN-CLASS INHIBITOR FOR THE MASTER ONCOREGULATOR HNRNP K IN MULTIPLE MYELOMA

Background: Haematological malignancies constitute a plethora of different neoplasms including leukaemia, lymphomas and myeloma. Despite all of them having robust first-line treatments and a myriad of therapeutic alternatives, some remain incurable due to the resistance and refractoriness of tumour cells. The heterogeneous nuclear ribonucleoprotein K, hnRNP K, is a master oncoregulator that binds C-rich tracks of nucleic acids and is implicated in multiple biological functions such as splicing, polyadenylation and translation. Indeed, hnRNP K contributes to treatment resistance and poor outcomes in haematological malignancies. Compared to other hnRNPs, hnRNP K has the unique structural characteristic of containing three K-homology (KH) domains that allows it to bind both ssDNA and RNA sequences, as well as a K-interactive (KI) region that binds to multiple critical proteins such as Src, Fyn or Lyn amongst others. Interestingly, hnRNP K regulates the p53/p21 and c-Myc pathways, and our group has characterised hnRNP K as a driver of leukaemia so far, thus proving its role in haematological neoplasms. Aims: We aim to develop a first-in-class inhibitor for hnRNP K and test its efficacy in circumventing resistance of haematological malignancies. Methods: We have produced full-length (FL) hnRNP K protein in-house and verified that it was properly folded and could bind RUNX1 RNA and mRNA sequence-based ssDNA, using Tycho and fluorescence anisotropy technologies. We then carried out a high-throughput screen of over 7,000 small molecules (both FDA and non-FDA approved) using AlphaLISA technology and validated both chemically and biologically the inhibition of hnRNP K with those identified compounds. Lastly, we have performed phenotypical analysis of the effects of the hnRNP K inhibitors in Multiple Myeloma cell line AMO1 and L363 overexpressing hnRNP K through CRISPR synergistic activation mediator (SAM). Results: We have successfully produced and purified the FL hnRNP K protein, with stable inflexion temperatures (Ti) as measured by Tycho. Furthermore, we corroborated that it maintained its ssDNA and RNA binding abilities through fluorescence anisotropy. Lastly, we set up the AlphaLISA system to screen a library of over 7,000 small molecules and identified circa 10 interesting hits, from which we validated the 5 best based on their inhibition capacity and specificity of hnRNP K. We verified the chemical properties of these small molecules. Lastly, we confirmed that they had a biological effect of decreasing the levels of hnRNP K downstream pathways as well as inducing changes in viability dose-response curves, using Multiple Myeloma cells (AMO1 and L363) genetically modified with CRISPR/SAM technology to overexpress hnRNP K. Likewise, we investigated the absence of such biological effect in hnRNP K-Knock Out (KO) AMO1 and L363 cells, obtained by CRISPR-Cas9. Image:Summary/Conclusion: We have identified a first-in-class hnRNP K inhibitor that specifically binds and blocks hnRNP K in modified Multiple Myeloma cell lines. This constitutes a novel and promising therapeutic approach that could help overcome the current drug resistance in haematological neoplasms currently found in the clinic. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295), ISCIII Miguel Servet (CP19/00140) and IF-Marie Sklodowska Curie Actions grant (MAtChinG – 101027864).

Dissecting the Adaptive Response to Arginine Deprivation in Hodgkin Lymphoma

Background In Hodgkin Lymphoma (HL), neoplastic cells orchestrate an inflammatory microenvironment leading to sterile inflammation, T-cell anergy, and immune deficiency. Our group showed that in HL patients the aminoacid degrading enzyme Arginase-1 is increased, associated with poor outcome, and leads to arginine (Arg) deprivation. However, how the reduction of Arg in the extracellular milieu of the tumor microenvironment can contribute to neoplastic cell fitness is largely unknown.Aims To detect the adaptive response (via evaluation of global transcriptome and metabolome changes) in human HL cell lines exposed to Arg deprivation.Methods To better understand the impact of extra-cellular Arg1 deprivation on the metabolome of human cHL cells, four human cHL cell lines (L428, L540, HDMYZ and KM-H2) were individually cultured with customized complete media or lacking or Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments. After 48 hours of culture, the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by Illumina platform. Following normalization to DNA concentration, log transformation, and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups.Results While Arg deprivation did not affect cell viability but delayed cell cycle due to arrest in G2 phase in all tested cell lines, the effect of Arg deficiency on the cellular metabolome depended largely on the cell type examined with L428 and KMH2 cells having significantly changed metabolomes. Pyruvate was significantly higher in the KMH2 cells deprived of Arg compared to controls. Conversely, lactate was significantly lower, with increased levels of long-chain saturated fatty acids and long-chain polyunsaturated fatty acids (PUFAs). Taken together the metabolomics changes suggested that specific-amino acid deficiency can lead to an increase in free fatty acids synthases to preserve cytoplasmatic and mitochondrial membrane dynamics. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione in KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine).Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response (UPR, including XBP1, EIF2S1, EIF4A2, EIF4A3, ATF3, ATF4, DDIT4, EDEM1, GADD45B, SQSTM1, HMOX), NF-kB response to TNF (including RAF1, TNF, LIF, NKBIA, SGK1, BIRC3, ICAM1, BCL6, IL6, RELA, CDKN1A), p53 pathway and networks (including CDKN2B, STOM, TRAF4, RRAD, SESN1, FOXO3, SERPINB5, JAG2) and proteosome degradation (HSPA4, PSMD11, PSMD13, PSMD2, PSMA5, PSMA7, PSMC4), with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation.All lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, suggesting that early response to arginine deprivation includes modulation of UFMylation pathway, the most recently discovered post-translational protein modification system, whose biological function is largely unknown.Conclusions The removal of Arg from L428 and KMH2 resulted in changes in the specific-amino acid-related metabolites. The adaptive response to Arg-depleted environment increases oxidative stress and promotes a shift in glucose use in the attempt to preserve mitochondrial function. The cell-cycle arrest in G2, the increase of pyruvate availability and the upregulation of proteasome function via upregulation of the UFMylation pathway suggest the dependency of HL cell lines on mitochondrial function integrity. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. DisclosuresMartinelli: Stemline Therapeutics: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Abbvie: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy. Di Raimondo: Pfizer: Honoraria; Jazz Pharmaceutical: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Mitochondrial Electron Transport Chain Inhibition Promotes Resistance to Proteasome Inhibitors in Multiple Myeloma

INTRODUCTIONProteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax.METHODSWe have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15).RESULTSWe find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy.CONCLUSIONWe show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. DisclosuresLonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

P-091: Genome-wide CRISPR/Cas9 screening identifies proteasome-related specific vulnerabilities as potential treatment options of proteasome inhibitor-resistant multiple myeloma

P-091: Genome-wide CRISPR/Cas9 screening identifies proteasome-related specific vulnerabilities as potential treatment options of proteasome inhibitor-resistant multiple myeloma

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Abstract 1478: TP-5809, a novel TNK1 inhibitor, suppresses TNK1 dependent signaling and tumor growth in a preclinical model of Hodgkin's lymphoma

Abstract TNK1 (thirty-eight-negative kinase 1) is an ACK non-receptor tyrosine kinase expressed during developmental hematopoiesis. Expression in adult tissues has been implicated in hematologic malignancies and in mediation of cellular stress and apoptosis. A truncating mutation leading to overexpression and hyperactivation of TNK1 drives growth and proliferation of L540 cells, a Hodgkin's lymphoma-derived cell line. TNK1 knockdown is known to reduce phosphorylation of downstream effectors STAT3 and STAT5 in L540 cells, to sensitize cancer cells to stress. However, there are no specific TNK1 inhibitors currently approved for use in hematological malignancies. We propose that pharmacological inhibition of TNK1 phosphorylation can suppress TNK1-driven pathology in preclinical tumor models. TP-5809 is a novel small molecule that potently inhibits TNK1 with an IC50 of 1.5 nM in a biochemical kinase assay. TP-5809 shows cellular target engagement at low nanomolar concentrations (IC50 = 11.3 nM) using a TNK1 Nanobret assay in HEK293 cells. In addition, TP-5809 inhibits TNK1-dependent Ba/F3 cell line growth with an IC50 less than 10 nM. In cell culture viability assays, TP-5809 has highest potency against the L540 Hodgkin's lymphoma cell line that expresses truncated TNK1 (IC50 = 7.2 nM), with nearly 1000-fold higher potency compared to K562 cells that express full length TNK1 and are not dependent on TNK1 for growth (e.g. IC50 = 2800 nM). TP-5809 inhibits STAT5 phosphorylation in L540 cells with an IC50 of 1 nM, whereas no pSTAT5 inhibition is observed in K562 cells. Importantly, TP-5809 efficiently inhibits growth of L540 Hodgkin's lymphoma in a mouse xenograft model but has no effect on K562 tumor growth. Daily dosing with TP-5809 inhibits tumor growth by 65.5% at 25 mg/kg, and by 78.7% at 50 mg/kg in the L540 xenograft mouse model, with a concurrent dose and time-dependent STAT5 and TNK1 phosphorylation inhibition. Pharmacokinetic and toxicology studies with TP-5809 have shown that exposure at biologically relevant doses is well-tolerated in mice, rats, and dogs. Taken together, these data show that TP-5809 potently inhibits TNK1 kinase function in biochemical and cellular assays. The data also show that TP-5809 actively inhibits TNK1 function in a TNK1-dependent xenograft model. We conclude that TP-5809 is a biologically active TNK1 inhibitor, which could allow further exploration of TNK1 as a clinically relevant therapeutic target in lymphoma and/or other TNK1-dependent cancers. Targeting aberrant TNK1 phosphorylation using TP-5809 can potently and specifically inhibit pathology driven by hyperactive and mutant forms of TNK1. Citation Format: Tetyana V. Forostyan, Ethika Tyagi, Jason M. Foulks, Matthew Lalonde, Joshua L. Andersen, Adam Siddiqui, Clifford J. Whatcott, David Bearss, Steve Warner. TP-5809, a novel TNK1 inhibitor, suppresses TNK1 dependent signaling and tumor growth in a preclinical model of Hodgkin's lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1478.

Heparanase Expression Is Associated With Cancer Stem Cell Features and Radioresistance in Hodgkin's Lymphoma Cells.

Heparanase (HPSE) is relevant to therapy resistance in many malignancies yet is largely unstudied in Hodgkin's lymphoma. Here, we investigated links between HPSE, cancer stem cell (CSC) features and radioresistance in KM-H2 and L428 Hodgkin's lymphoma cells. Firstly, HPSE expression in unsorted and sorted CSCs was assessed. Post-irradiation, HPSE and CSC-related gene expression changes were then quantified. Clonogenic ability was investigated with and without artificial changes in HPSE expression pre and post irradiation. HPSE was highly expressed in L428 but barely present in KM-H2 cells. HPSE was overexpressed in sorted L428 CSCs. Irradiation induced HPSE and expression of CSC markers. High HPSE-expressing L428 cells showed higher clonogenic ability than low HPSE-expressing KM-H2 cells after irradiation. Down-regulation of HPSE in L428 cells reduced their clonogenic capability post-radiation, whilst overexpression of HPSE in KM-H2 cells increased colony formation. HPSE expression is associated with CSC features and contributes to radioresistance in Hodgkin's lymphoma cells.

Glutamine Catabolism Maintains MCL-1 Expression in Glucose-Deprived Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy accounting for 11,000 deaths annually in the US with the majority succumbing to disease due to the development of resistance. A key component to the development of resistance lies in ineffective engagement of the BCL-2 family of apoptosis regulators. We and others have demonstrated that glucose maintains levels of a key resistance-promoting BCL-2 family member, myeloid cell leukemia factor 1 (MCL-1). Expression of MCL-1 promotes resistance of MM to a wide range of therapeutics. MM, like other cancers, exhibits elevated glucose consumption associated with manifestation of the Warburg effect. We previously reported that myeloma cells maintain the glycolytic phenotype by achieving constitutive plasma membrane localization of GLUT4, and GLUT4 inhibition or glucose deprivation suppressed MCL-1 expression. In this study, we further investigated the mechanism underlying the suppression of MCL-1 expression upon glucose deprivation.L363 and KMS18 cell lines are sensitive to glucose deprivation, exhibiting significant cytotoxicity upon glucose deprivation and demonstrating rapid reduction of MCL-1 protein in contrast to JJN3 and KMS11 cells that are resistant to glucose deprivation. Knockdown of MCL-1 in JJN3 dramatically induced apoptosis while overexpression of MCL-1 in L363 significantly reversed cell death upon glucose deprivation, supporting a role for MCL-1 suppression in mediating glucose-deprivation elicited cell death. We next proceeded to investigate the metabolic pathways contributing to the persistence of MCL-1 in the glucose-insensitive lines. Since glutamine is an important carbon source that can compensate for glucose deprivation, we examined glutamine uptake in L363 and JJN3 cells. Evaluation of 3H-glutamine uptake revealed that JJN3 cells exhibit higher glutamine uptake than L363 cells. In addition, we performed 13C-based metabolic flux analysis in order to identify metabolites that may be contributing to the maintenance of MCL-1 in glucose-deprived cells. Interestingly the level of glutathione (GSH) was much higher in JJN3 than in L363 that was maintained even upon glucose deprivation. Since GSH is a major cellular reactive oxygen species (ROS) scavenger, we evaluated levels of ROS using a cyto-roGFP construct and found that it was significantly elevated upon glucose withdrawal in L363 but not in JJN3. In addition, treating glucose deprived L363 with cell-permeant GSH monoethyl ester (GSH-MEE) rescued MCL-1 expression, further supporting a role for ROS in suppressing MCL-1 expression.Glutamine uptake evaluation revealed that glutamine consumption was maintained upon glucose withdrawal in both JJN3 and L363, so we were then interested in the TCA cycle-related differences in glutamine metabolism between JJN3 and L363. The level of pyruvate dehydrogenase alpha 1 (PDHA1) phosphorylation decreased upon glucose deprivation only in JJN3 cells, which activates PDHA1 and allows pyruvate to flow into the TCA cycle. In accordance with this, treatment of JJN3 with dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase and therefore activates PDHA1, restored MCL-1 expression and prevented cell death of L363 during glucose deprivation. To study how TCA cycle helps maintain the GSH level, glutamate dehydrogenase (GDH) knockdown cells were generated because GDH catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and glutamate is the precursor for GSH. Interestingly, in GDH knockdown L363 cells, treatment of DCA was not able to reverse MCL-1 protein level upon glucose deprivation. Taken together, our observations suggest that glucose-deprived resistant cells sustain higher levels of GSH via greater glutamine uptake, thereby scavenging ROS to sustain cell viability; while sensitive cells cannot make GSH effectively, leading to increased ROS level, decreased MCL-1 expression, and ultimately, apoptosis.In sum, we have discovered that targeting glucose entry elicits apoptosis via an induction of ROS leading to suppression of MCL-1. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and the need to further understand how glutamine metabolism and the TCA cycle contribute to maintaining MCL-1 expression. DisclosuresNo relevant conflicts of interest to declare.