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B-cell maturation antigen (BCMA) is selectively expressed as a surface protein on plasma cells and is a validated target for multiple myeloma (MM). A soluble form of BCMA (sBCMA) is elevated in the sera of patients with MM. The bispecific antibodies teclistamab (BCMA×CD3) and talquetamab (G protein-coupled receptor family C group 5 member D [GPRC5D]×CD3) are in clinical development as therapies for MM. Using data from patients with relapsed/refractory MM (N=300) who participated in phase 1 studies of teclistamab (NCT03145181) or talquetamab (NCT03399799), baseline serum sBCMA levels were quantitatively analyzed relative to response to treatment, percentage of bone marrow plasma cells (BMPCs), and cytogenetic risk. By cycle 3 day 1 of treatment, sBCMA levels declined from baseline in 50 (88%) of 57 responders and 49 (98%) of 50 responders to teclistamab and talquetamab, respectively. Depth of response correlated with the magnitude of sBCMA reduction. In contrast, sBCMA increased in 33 (80%) of 41 patients and 24 (49%) of 49 patients who did not respond to teclistamab or talquetamab, respectively. Baseline sBCMA levels correlated with the percentage of BMPC; patients with extramedullary plasmacytomas who had low levels of BMPC (≤10%) tended to have high baseline sBCMA levels (≥400 ng/mL), based on limited data. Baseline sBCMA levels and changes in sBCMA levels at cycle 3 day 1 were similar in patients with high- and standard-risk cytogenetics treated with teclistamab or talquetamab. These results support the use of sBCMA as a potential surrogate marker of tumor burden and treatment response in MM.

Background B-cell maturation antigen (BCMA, CD269), a single transmembrane protein, can be shed as a soluble 6 kilodalton protein fragment (sBCMA) upon cleavage at the transmembrane domain by γ-secretase. Given its selective expression in the B-cell lineage, BCMA is a validated target for multiple myeloma. Teclistamab and talquetamab are CD3 bispecific antibodies developed to recruit CD3+ T cells to BCMA+ or G protein-coupled receptor family C group 5 member D (GPRC5D)+ multiple myeloma (MM) cells, respectively. Levels of sBCMA after teclistamab or talquetamab treatment were evaluated in patients with relapsed and/or refractory MM (RRMM). Methods Serum samples from patients with RRMM in teclistamab and talquetamab phase 1 studies (64007957MMY1001 and 64407564MMY1001, respectively) were collected at various timepoints between baseline and cycle 4 or end of treatment and analyzed for sBCMA by an electrochemiluminescence ligand binding assay. Quantitative analyses of sBCMA data were conducted with reference to a patient’s tumor burden and response, and to pharmacokinetic data. Results Teclistamab and talquetamab modulated levels of sBCMA in patients with high (≥50%) and low ( Conclusions Changes in the levels of sBCMA upon treatment with teclistamab and talquetamab correlated with clinical activity, supporting further clinical development. The findings support BCMA as a potential surrogate marker of myeloma tumor burden and as a valuable marker for response in patients with MM.

Introduction: Daratumumab (DARA)-based regimens are effective and well tolerated in both newly diagnosed and relapsed/refractory multiple myeloma (RRMM) patients. However, the prognosis for patients who have become refractory to proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs) and DARA is poor. A better understanding of determinants of response and mechanisms of resistance to DARA may lead to new rationally designed treatment strategies. We therefore characterized the effect of DARA on the immune system in MM patients treated with DARA monotherapy in part A of the DARA-ATRA study (ClinicalTrials.gov NCT02751255). Methods: In part A of this prospective multicenter phase 2 trial, 63 DARA-naïve patients were treated with DARA monotherapy (16 mg/kg; approved schedule). Median number of prior lines of treatment was 4 (range 2-11), all patients were previously exposed to lenalidomide and a PI; 89% was refractory to an IMiD, 71% to a PI and 67% to both IMiD and PI. Bone marrow (BM) aspirates obtained at baseline (BL) and at progression (PD; primary refractory, or acquired resistance after prior response) were subjected to deep immune profiling using 28 surface- and intracellular proteins measured by flow cytometry (BL n=51, PD n=47). In a subset of these patients (BL n=39, PD n=33) mass cytometry (CyTOF) was also performed profiling 39 proteins. In addition, peripheral blood (PB) samples obtained at BL (n=44) and PD (n=37) were analyzed using CyTOF. Computational analyses of flow data were performed using UMAP and FlowSOM; CyTOF data were analyzed using SPADE and Freeviz. Statistical analyses included Wilcoxon rank-sum and signed-rank tests, Generalized Linear Mixed Models and ANOVA. Results: A partial response or better was achieved in 41% of patients treated with DARA monotherapy. We compared immune profiles of responding and non-responding (primary-refractory) patients. At BL, the percentages of MM-, T-, B- and NK cells in BM were similar between both groups. However, NK cells of non-responding patients had a lower proportion of CD16 + (P=0.029), and a higher proportion of TIM-3 + (P=0.010) and HLA-DR + (P=0.043) NK cells, suggesting an exhausted phenotype. Non-responders also had a higher proportion of TIM-3 + CD4 + (P=0.022) and TIM-3 + CD8 + T-cells (P=0.004), and a higher proportion of TIM-3 + regulatory T-cells (Tregs) (P=0.042). A higher proportion of TIM-3 + -NK cells, -T-cells, or -Tregs in BM was also associated with poor progression-free and overall survival (PFS; OS). In addition, clinical characteristics associated with poor PFS such as LDH (P=0.016), extramedullary disease (P=0.001) and (R-)ISS stage III (P=0.015) were associated with an increased number of phenotypically exhausted NK- and T-cells. Similar to prior studies, DARA treatment resulted in reduced levels of CD38 on all immune cell subsets and a marked decrease in Tregs, regulatory B cells (Bregs) and NK cells in both BM and PB. Upon acquired resistance, remaining NK cells displayed higher proportions of TIM-3 + (P=0.022), HLA-DR + (P=0.0007) and LAG-3 + (P=0.011), and lower proportions of CD16 + (P=0.002) (Figure 1), compatible with an exhausted phenotype. Furthermore, disease progression was associated with an increase in CD4 + and CD8 + terminally differentiated effector memory T-cells and a decrease in CD4 + and CD8 + central memory T-cells in BM. There was no significant change in the proportion of T-cells expressing immune checkpoint molecules. CyTOF analysis of BM and PB samples confirmed flow cytometric findings. Furthermore, data-driven analysis identified immune profiles specific to progression, including a significant decrease in the fraction of Granzyme B + NK cells, and increase in the fraction of Granzyme B + Bregs at the time of progression. Based on NK cell depletion and -exhaustion observed at PD, we hypothesized that NK cell repletion may restore DARA sensitivity. Indeed, in ex vivo experiments we show that DARA-resistance can be overcome by addition of healthy donor derived NK cells to MM cells obtained from DARA-refractory patients. Conclusion: Here we show that an increased proportion of NK cells with an exhausted phenotype is associated with primary and acquired DARA-resistance, which is in line with the important role of NK cells in DARA-mediated tumor cell elimination ex vivo. Future DARA-based treatment strategies may benefit from reinvigorating NK cells and restoring their cytotoxic capacities. Figure 1 Figure 1. Disclosures Zweegman: Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Minnema: Celgene: Other: Travel expenses; Alnylam: Consultancy; Cilag: Consultancy; Janssen: Consultancy; Kite/Gilead: Consultancy; BMS: Consultancy. Broyl: Celgene/BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Kersten: Kite/Gilead: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Roche: Honoraria; Takeda: Consultancy; BMS/Celgene: Consultancy; Miltenyi Biotech: Consultancy. Krevvata: Janssen: Current Employment. Casneuf: Janssen: Current Employment. Abraham: Janssen: Current Employment. Verona: Janssen: Current Employment. Smets: Janssen: Current Employment. Vanhoof: Janssen: Current Employment. Cortes-Selva: Janssen: Current Employment. van Steenbergen: Biolizard working for Janssen: Current Employment. Vieyra: Janssen: Current Employment. Sonneveld: Karyopharm: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Mutis: Janssen: Honoraria; Genmab: Research Funding; Takeda: Research Funding; Novartis: Research Funding; ONK Therapeutics: Research Funding. van de Donk: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

8047 Background: B-cell maturation antigen (BCMA, CD269) is a single transmembrane protein that is selectively expressed in the B-cell lineage and is a validated target for multiple myeloma. BCMA exists as both surface protein and as a free soluble form (sBCMA). γ-secretase activity at the transmembrane domain leads to a shed BCMA protein fragment of approximately 6 kilodalton that can exist as free circulating sBCMA in blood. Teclistamab and talquetamab are CD3 bispecific antibodies that have been developed to recruit CD3+ T-cells to BCMA+ or GPRC5D+ multiple myeloma (MM) cells, respectively. The objective of this work was to evaluate sBCMA in relapsed and/or refractory MM patients in response to treatment with teclistamab or talquetamab. Methods: Serum samples from relapsed and/or refractory MM patients in teclistamab and talquetamab phase 1 studies (64007957MMY1001 and 64407564MMY1001) were collected (at various timepoints between baseline and cycle 4 or end of treatment) and analyzed for sBCMA by an electrochemiluminescence ligand binding assay. Soluble BCMA data were quantitatively analyzed in reference to patient’s tumor burden and response, as well as pharmacokinetic data. Results: Teclistamab and talquetamab modulated levels of sBCMA in patients with high ( ≥ 50%) and low ( < 50%) frequency of tumor plasma cells (TPCs), as well as in high and low risk cytogenetic groups. In cycle 3, majority of the responders had reduction in sBCMA [88% (50 out of 57) for teclistamab and 98% (49 out of 50) for talquetamab] compared to baseline. On the contrary, non-responders (progressive disease, stable disease, or minimal response) seemed to show an increase in sBCMA [80% (33 out of 41) for teclistamab and 49% (24 out of 49) for talquetamab] from baseline. Patients with deep responses tend to have higher magnitude of sBCMA reduction compared to others. Based on few patients who responded to teclistamab or talquetamab and then relapsed, sBCMA seemed to have an initial reduction followed by an increase in the levels. Soluble BCMA corelated with % bone marrow TPCs. Majority of patients with plasmacytoma (limited data) seemed to have high sBCMA; suggesting sBCMA could be a comprehensive marker for tumor burden. Teclistamab preliminary population pharmacokinetic analysis showed that sBCMA did not appear to impact teclistamab exposure, suggesting that sBCMA was not acting as a sink for teclistamab. Conclusions: Teclistamab and talquetamab induced changes in levels of sBCMA that correlated with clinical activity, further supporting clinical development of these bispecific antibodies. Lastly, the results support that sBCMA is a potential surrogate marker of myeloma tumor burden, and as a valuable marker for response in MM patients. Clinical trial information: NCT03145181 and NCT03399799.

Previous studies have shown that UV-induced binding of p21(WAF1) to PCNA through the PCNA-interacting protein (PIP) domain in p21(WAF1) promotes a switch from DNA replication to DNA repair by altering the PCNA protein complex. Here we show that the p33(ING1b) isoform of the ING1 candidate tumour suppressor contains a PIP domain. UV rapidly induces p33(ING1b) to bind PCNA competitively through this domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG exo/endonucleases, and DNA methyltransferase. Interaction of p33(ING1b) with PCNA occurs between a significant proportion of ING1 and PCNA, increases more than tenfold in response to UV and is specifically inhibited by overexpression of p21(WAF1), but not by p16(MTS1), which has no PIP sequence. In contrast to wild-type p33(ING1b), ING1 PIP mutants that do not bind PCNA do not induce apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for this PCNA-p33(ING1b) interaction in eliminating UV-damaged cells through programmed cell death. These data indicate that ING1 competitively binds PCNA through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex.

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12–48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.

Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.

Clinical and experimental evidence suggest that the p33ING1b candidate tumor suppressor functionally cooperates with p53 in controlling biochemical and biological functions. Because p53 is frequently mutated in brain tumors and the ING1 locus maps to a site of which the loss is associated with gliomas, we analyzed the mutation and expression profiles of ING1B in human brain tumors. Here we present the first report of ING1 expression and mutation analyses in human brain tumor samples and malignant glioma cell lines.Expression and mutation analyses of ING1B together with subcellular localization studies of ING1 proteins were performed on 29 brain tumor specimens and 6 human glioma cell lines.A single point mutation (3.5%) was detected in the 29 brain tumor specimens analyzed. This missense mutation occurred in a sequence reported previously to confer nuclear translocation properties to p33ING1b. Interestingly, overexpression and subcellular mislocalization of p33ING1b were observed in all 29 of the brain tumor specimens and some glioma cell lines. In tumor samples, ING1 proteins aberrantly localized to the cytoplasm, and to a lesser extent, to the nucleus of glioma cells.Our data indicate that although mutations of ING1 seem to be infrequent in human brain tumors, deregulated expression and mislocalization of ING1 proteins, particularly the p33ING1b isoform, are common events in gliomas and glioblastomas.

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.

Recently, several novel human ING1 isoforms have been cloned. However,the biochemical functions and the involvement of these proteins in apoptosis remain uncharacterized. We have examined the apoptotic effects and biochemical functions of the two major human ING1 isoforms p47(ING1a) and p33(ING1b) in young and senescent human diploid fibroblasts induced to enter into apoptosis by diverse treatments. We have found that ING1 displayed isoform-, stimulus- and cell age-dependent apoptotic properties. We present evidence indicating that ING1 proteins bind to chromatin and are regulated in a manner related to their apoptotic properties. In agreement with previous reports, we have found that only young but not senescent fibroblasts were able to enter into apoptosis induced by growth factor deprivation. This effect was accompanied by up-regulation of endogenous p33(ING1b). Ectopic up-regulation of p33(ING1b), but not p47(ING1a), also induced apoptosis and sensitized young but not senescent cells to UV irradiation and hydrogen peroxide-mediated apoptosis. Cotransfection of p33(ING1b) and the tumor suppressor p53 increased the percentage of apoptotic cells yielded by either of these two proteins alone, in agreement with data from tumor cell models. Finally, we found that the chromatin binding affinity of p33(ING1b) was increased in senescent cells, which were resistant to apoptosis. Together, these data support the idea that the apoptotic functions of ING1 may be exerted by chromatin-related functions that are subject to cell age-dependent mechanisms of regulation.

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33ING1b, but not of p47ING1a, induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47ING1a inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33ING1b affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT·ING1·PCNA protein complexes.