Cogen, Davitte

Abstract Background Some patients with inflammatory bowel disease (IBD) on immunosuppressive therapies may have a blunted response to certain vaccines, including the messenger RNA (mRNA) coronavirus disease 2019 (COVID-19) vaccines. However, few studies have evaluated the cell-mediated immune response (CMIR), which is critical to host defense after COVID-19 infection. The aim of this study was to evaluate the humoral immune response and CMIR after mRNA COVID-19 vaccination in patients with IBD. Methods This prospective study (HERCULES [HumoRal and CellULar initial and Sustained immunogenicity in patients with IBD] study) evaluated humoral immune response and CMIR after completion of 2 doses of mRNA COVID-19 vaccines in 158 IBD patients and 20 healthy control (HC) subjects. The primary outcome was the CMIR to mRNA COVID-19 vaccines in patients with IBD. The secondary outcomes were a comparison of (1) the CMIR in patients with IBD and HC subjects, (2) CMIR and humoral immune response in all participants, and (3) correlation between CMIR and humoral immune response. Results The majority (89%) of patients with IBD developed a CMIR, which was not different vs HC subjects (94%) (P = .6667). There was no significant difference (P = .5488) in CMIR between immunocompetent (median 255 [interquartile range, 146-958] spike T cells per million peripheral blood mononuclear cells) and immunosuppressed patients (median 377 [interquartile range, 123-1440]). There was no correlation between humoral and cell-mediated immunity after vaccination (P = .5215). In univariable analysis, anti-tumor necrosis factor therapy was associated with a higher CMIRs (P = .02) and confirmed in a multivariable model (P = .02). No other variables were associated with CMIR. Conclusions Most patients with IBD achieved CMIR to a COVID-19 vaccine. Future studies are needed evaluating sustained CMIR and clinical outcomes.

Abstract Background: Aberrant B-cell receptor (BCR) and MYD88 signaling cooperatively drive Waldenstrom macroglobulinemia (WM) tumor cell survival. To sustain BCR-driven growth and maintain optimal cellular homeostasis, WM cells upregulate proteasome function. We have recently described the role of two distinct 19S proteasome-associated deubiquitinating enzymes (DUBs), USP14 and UCHL5, in maintaining WM-cell viability. Pharmacologic inhibition with b-AP15 (commercial compound) results in WM-cell death and accumulation of high molecular weight ubiquitinated protein conjugates; an event that is phenocopied by siRNA knockdown of USP14 and UCHL5. VLX1570 is a lead-optimized clinical grade analog of b-AP15 with enhanced solubility, stability and target residence time. We investigated the effect of VLX1570 on BCR components, WM cell survival proteins and its in vivo activity in a human xenograft model of WM. Materials: VLX1570 was obtained from Vivolux AB and bortezomib from Sellekchem. Human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1) and their drug-resistant derivatives were used (total n=12). Bone marrow aspirates from WM-patients and from healthy donors were obtained from the Mayo Clinic biospecimens core. PBMCs from healthy donors were used as comparators where needed. NOD/SCID mice were obtained from Taconic Labs and used for profiling the in vivo properties of VLX1570. Results: We first examined for USP14/UCHL5 protein expression by immunohistochemical analysis in primary tumor tissue from WM patients and compared it to healthy donor cells. WM patient-derived cells exhibited greater staining intensity of both DUBs. Preferential binding of VLX1570 to USP14 and UCHL5 in WM cells was probed in a WM cell using an enzyme-based competition assay (HA-Ub-Vs), which demonstrated time-dependent decrease in labeled USP14 and UCHL5 after treatment with VLX1570. In drug-resistant (bortezomib or ibrutinib-resistant) WM cells, VLX1570 induced >50% tumor-specific apoptosis and displayed synergistic activity with ibrutinib (median CI: 0.5). In addition to ER stress markers, we had previously observed that gene expression of BCR-signalosome components were reduced in VLX1570-treated WM cells (NanoString platform) and this was substantiated at the protein level where p-SYK, SYK and BTK decreased in a time-dependent manner. BCR end-effectors were probed in response to VLX1570 and a decrease in nuclear translocation of NFkB (p65) was noted. USP14 has been shown to control the availability of the chemokine receptor CXCR4, which regulates homing of WM cells to bone marrow. After VLX1570 treatment, we noted a significant decrease in CXCR4 surface receptor expression in all WM cell lines tested and primary WM cells. Lastly, to determine the in vivo anti-WM effects of VLX1570, we injected NOD/SCID mice (xenotransplanted with RPCI-WM1-Luc cells) with VLX1570 (I.P. route, 4.4mg/kg every other day) and measured tumor burden along with pharmacodynamic indicators. VLX1570-treated mice (n=7) demonstrated significantly lower tumor volumes by caliper measurement and bioluminescent signal (p=0.01 - 0.003) as well as reduced serum human-IgM, compared to vehicle-treated mice (n=7). Importantly, mice treated with VLX1570 survived longer vs. vehicle-treated mice (median survival, 62 vs. 44 days, respectively, p=0.0008). No unforeseen toxicities were noted and weight of the mice in vehicle vs. VLX1570-treated mice did not significantly change. IHC analysis in mice tumor tissues demonstrated reduced CXCR4, human-IgM, Ki67 and increased cleaved caspase-3 and ubiquitinated (k48-linked) protein level, the latter serving as a more specific indicator of USP14/UCHL5 inhibition. Conclusions: We provide substantial in vitro and in vivo evidence on the anti-WM effects of a novel first-in-class clinical grade USP14/UCHL5 inhibitor, VLX1570. In addition to anticipated induction of ER stress machinery, we describe for the first time VLX1570-mediated modulation of BCR components and WM cell survival proteins as well as demonstrate its robust activity in an aggressive human xenograft model of WM. These data provide the rationale for a clinical trial of single agent VLX1570 in patients with symptomatic WM. Disclosures Martin: Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Gullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background: Current tumor profiling analytics provide some insight into the various molecular abnormalities and their individual consequences on oncogenic signaling. However, these analyses are limited by their lack of integration where the combined effect of individual mutations, gene copy number variations and chromosomal aberrations are not consolidated to create the global molecular architecture that supports neoplastic growth, particularly in the context of drug resistance. Consequentially, identities of the preferential oncogenic pathway(s) tumor cells employ to oppose the effects of targeted therapies remain cryptic and unactionable. Here we present a simulation-based method, which not only replicates the molecular architecture of ibrutinib-resistant Waldenstroms Macroglobulinemia (WM, for which ibrutinib is the only FDA-approved agent) in silico, but also predicts cell sensitivity towards existing drugs, which we validated experimentally for potential clinical translation. Materials: We used the newly established human WM cell line, RPCI-WM1/IR, as a surrogate model of ibrutinib-refractory WM. Genomic data including whole exome sequencing (WES) and copy number analysis (CNA) was utilized for the creation of an avatar of RPCI-WM1/IR, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were nominated. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of the WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. Results: Several genomic aberrations were used to create the RPCI-WM1/IR simulation avatar. Functional activity (based on mutation or copy number alteration) of several ibrutinib targets or transcription factors associated with BTK activity such as FYN, SP1, BMX and FRK were predicted to be lost. Increased expression of CAV1, which also inhibits BTK mediated signaling, was increased. An increase in CSNK2B, which activates PU.1- a transcriptional target of BTK, was also observed. Of note, no CXCR4 mutations, which have been shown to impact ibrutinib response, were observed. Next, the cytotoxic potential of over 150 FDA approved drug (and some in experimental stages) were simulated individually and in combination on the RPCI-WM1/IR avatar. In silico modeling predicted aberrant activity of aurora kinase A (AURKA) and its associated signaling partners, which could be disrupted with the (AURKA) inhibitor, tozasertib. AURKA activation was predicted as upregulated due to alterations in several genes: RASA1 loss and SOS1 increase --> increased ERK --> increased ETS1 --> increased AURKA. High beta-catenin signaling (high CTNNB1 and FZD1/4 and low AXIN1 and GSK3B) was also shown to increase AURKA. The simulation predictions were experimentally validated in vitro where AURKA inhibition with tozasertib significantly inhibited proliferation of RPCI-WM1/IR cells (IC50~14nM) as well as inducing apoptosis (48hr, 20nM treatment) and cell-cycle arrest. Conclusions: Our data demonstrates the potential of in silico modeling in predicting novel drug targets, allowing guidance in 1.) Delineating operational oncogenic circuits in an ibrutinib-resistant state by reanimation of the molecular architecture in silico, 2.) Calculating the impact of individual genomic abnormalities and their collective influence on maintaining tumor survival and 3.) Performing a rapid in-silico drug-sensitivity screen directed by the pathway analyses, which can be validated experimentally using standard assays. This novel approach holds tremendous potential in creating highly personalized therapies for ibrutinib-refractory WM patients based on unique genetic signatures. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

Abstract Background: The clinical impact of ibrutinib is irrefutable and has been hailed as a revolutionary treatment for several cancers, including mantle cell lymphoma (MCL). Despite high response rates noted with ibrutinib therapy in MCL (ORR, 68%), complete responses are low and durability of remission is suboptimal in MCL vs. CLL or Waldenstrom macroglobulinemia (WM) patients (Cheah et al Ann Oncol 2015). This relative lack of efficacy may be due to MCL cells having a higher proliferation rate and being less indolent in nature than CLL or WM cells. Tumor cells with a high proliferation index rely on optimally functioning protein metabolism/homeostasis systems. In MCL, this observation has been fortuitously exploited with the use of bortezomib, a 20S proteasome inhibitor. Indeed the ubiquitin proteasome system is a validated target and consists of several individual components whose function in MCL has yet to be mapped out. We have recently reported that inhibition of the 19S proteasomal deubiquitinating enzymes (DUBs), USP14 and UCHL5; with the small molecule inhibitor b-AP15 is lethal to drug-resistant WM tumor cells. This intriguing observation, prompted us to define the role of USP14/UCHL5 and their inhibition in a diverse panel of B-cell lymphoma cell lines, including MCL models as well as ibrutinib-resistant MCL cells. Materials: b-AP15 (and its clinical grade analog, VLX1570) was obtained from Vivolux AB and bortezomib from Sellekchem. Human NHL (DLBCL, MCL, Burkitt), Hodgkin lymphoma (HL), multiple myeloma (MM) cell lines as well as established drug-resistant subclones were used in drug-sensitivity screening experiments (total n=25). Results: USP14 and UCHL5 mRNA expression was queried in the CCLE database (Broad-Novartis) and showed highest expression in plasma cell and B-lymphoid tumor cell types. This was also observed at the protein level, wherein analysis of The Human Protein Atlas database revealed moderate to high intensity staining for USP14 and UCHL5 localizing to the cytoplasmic/membranous and nuclear compartments in high-grade NHL tissues. Next, we examined B-lymphoid tumor cell sensitivity to pharmacologic inhibition of USP14/UCHL5 with either b-AP15 or VLX1570 (72hr fluorescein diacetate hydrolysis assay or CellTiter Glo). We observed that MCL cells, including those resistant to ibrutinib (Jeko/IR cell line) were highly sensitive to VLX1570 (median IC50 12 - 25nM). In contrast, MM cells were not as sensitive (IC50's ranging between 58 - 90nM). Nine DLBCL cell lines exposed to equivalent time/concentrations of b-AP15 showed a median IC50 of 311nM (range, 70 - 910nM) and 3 HL models exhibited an IC50 of 531nM (range, 420 - 783nM). No difference in sensitivity to VLX1570 was noted between ABC-type vs. GCB-type DLBCL models. These functional results aligned with prior genomic inquiry where USP14 and UCHL5 gene expression was noted to be higher in MCL cell lines compared to DLBCL and non-hematologic cancer models analyzed (FDR=0.0019, p=0.001), suggesting their more central role in maintaining MCL tumor viability. Indeed MCL tumor survival (particularly ibrutinib-resistant cells) appears linked to USP14/UCHL5 function as treatment of Jeko/IR cells with VLX1570 (250nM, 12hrs) caused marked apoptosis (71.8% annexin-V+ staining) and cleavage of PARP-1. Moreover, the combination of ibrutinib and VLX1570 synergistically induced MCL cell death (median CI: 0.3). Mechanistic studies demonstrated that VLX1570 modulates several proteins critical for MCL survival including cyclin-D1, Myc and CXCR4/CD184. Reduction in cyclin-D1 was also noted to coincide with cell cycle arrest of VLX1570-treated MCL cells. Altogether, these findings suggest that VLX1570 modulates critical cellular signaling pathways utilized by MCL tumor cells, particularly those that are involved in resistance to ibrutinib. Conclusions: MCL remains a challenging malignancy to treat with no effective treatments for ibrutinib-relapsed disease. Our data supports the role of the proteasome-associated DUBs, USP14 and UCHL5, as critical factors involved in ibrutinib-resistant MCL tumor survival. VLX1570 is a potentially effective agent for the treatment of ibrutinib-resistant MCL. Disclosures Gullbo: Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.