Guilatco, Angelo Jose

Abstract Background: Patients (pts) with myeloma (MM) receiving CAR-T therapy have usually been exposed to many prior therapies. Melphalan (HDM) and other chemotherapies can induce cellular senescence. It has recently been shown that MM cells can also exhibit features of therapy-induced senescence (TIS). TIS tumor cells are associated with resistance to therapy and relapse. To investigate the presence of TIS MM cells prior to CAR-T and association with response, we examined the transcriptome and phenotype of MM prior to CAR-T. Method: We used scRNA-seq data from bone marrow (BM) MM cells: Boiarsky, et al. (GSE193531, dbGaP: phs001323.v3.p1) newly diagnosed MM (NDMM) pts; Dhodapkar, et al. (GSE210079), pre- and post CAR-T; pts who received CAR-T at Mayo Clinic, pre- (N=10) and post- (N=2). To evaluate TIS, single-cell gene set enrichment analysis (scGSEA) for several gene sets (PMID: 40164720): SenUp (Senescence Upregulated), SenGA (Senescence Growth Arrest), and SCAPs (Senescent Cell Anti-apoptosis Pathways); and a Plasma Cell Senescence (PCSen) gene set (GSE5900, GSE47552). To assess TIS and myeloid phenotype, BM MM cells (CD138+) were stained for CD14, CD16, KLRG1 and GPNMB and analyzed by CytoFlex and Kaluza. Results: In the integrated dataset, scGSEA showed significantly higher enrichment of PCSen, SenUp, SenGA, and SCAPs in late-stage MM cells (both pre- and post-CAR-T) compared to NDMM and healthy donors (p<0.05, Kruskal-Wallis with Dunn’s posttest). Conversely, the Normal gene set was significantly enriched in healthy plasma cells relative to MM (p<0.05). Expression of CDKN2A, which encodes the senescence marker p16, was also increased in late-stage MM relative to NDMM or healthy plasma cells. Notably, in our newly generated scRNA-seq samples collected within 6 months before CAR-T, a significant inverse correlation between senescence marker expression and progression free survival (PFS) post-CAR-T was seen, suggesting that MM cells exhibiting TIS may be more resistant to T cell mediated cytotoxicity. We found that 62.5% (25/40) of pts had >1% MM cells that were CD14+. Among them, the median CD14+ MM cells were 5.5% (1-71%); and median CD14+CD16+ MM cells were 2.14% (0.4-67%). Among 7 pts with available samples for KLRG1 and GPNMB assessment, 100% (7/7) had KLRG+ MM cells (median: 59% (24-99%)) and 57% (4/7) had GPNMB+ MM cells (median: 15% (3.9-30%)), and KLRG1+GPNMB+ MM cells (median: 9.6% (3.8-17%)). Conclusion: Our study shows that late-line MM cells exhibit transcriptional and phenotype profiles consistent with TIS. This senescent phenotype may contribute to resistance to immunotherapies, including CAR-T. Additional phenotype data is collected and will be presented. A phase II clinical trial is ongoing (NCT06940297) to assess for the combined treatment with senolytic drugs dasatinib and quercetin peri-CART to deepen clinical response. Citation Format: Sofia Arbelaez, Andre De Menezes Silva Corraes, Gabriel Alvares Borges, Chen Wu, Christoph Schaefers, Malvika Gupta, Zuoyi Shao, Kevin Regan, Rayaan Kamal, Angelo Jose Guilatco, Neal I. Sannuli, Ying Li, Taxiarchis Kourelis, Wilson Gonsalves, Moritz Binder, Melinda Tan, Nadine Abdallah, Megan Weivoda, Yi Lin. Myeloma cells with therapy-induced senescence-like phenotype are associated with resistance to CAR-T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6467.

Abstract Background: Therapy-induced senescence (TIS), a tumor cell state associated with distinct gene expression patterns and implicated in therapy resistance, may be activated in Multiple Myeloma (MM) patients (pt) by genotoxic chemotherapies, such as high-dose melphalan (HDM). We investigated whether TIS is activated in MM cells following chemotherapy and linked to resistance to T-cell directed therapies. Method: We integrated scRNA-seq data from healthy donors (CNTRL) and MM pts (newly-diagnosed (NDMM), pre/post-CAR-T) bone marrow plasma cells (PCs) using two public datasets (Boiarsky, et al.; Dhodapkar, et al.) and a newly generated pt dataset. Senescence was evaluated using scGSEA with published gene sets (SenUp, SenGA, and SCAPs). For in vitro MM cytotoxicity assay, human H929 MM cells were treated with vehicle (Veh) or HDM (3µM, IC90) for 6h and examined for TIS features. H929VEH or H929HDM were co-cultured with CNTRL T cells with or without teclistamab (Tec, 10 nM) or with day 14 PBMCs from MM pts post-CAR-T (E:T 5:1). CAR+ T cells were high among the day 14 PBMC (median±SD: 93.4±7.2% of CD3+ cells, n=6). Results: scGSEA revealed significantly higher enrichment of SenUp, SenGA, and SCAPs in late-stage MM cells compared to NDMM and CNTRL (p<0.05). Expression of CDKN2A, encoding the senescence marker p16, was also increased in late-stage MM relative to NDMM/CNTRL PCs. Of interest, cells enriched for senescence also exhibited enrichment for myeloid markers (TET2, CEBP family). Notably, in pre-CAR-T, increased baseline senescence or myeloid marker expression showed an inverse association with CAR-T progression-free survival (PFS), suggesting TIS confers resistance to T cell mediated cytotoxicity. This may be due to myeloid plasticity. TIS MM cells generated in vitro, H929HDM, exhibited a distinct morphology characterized by increased cell size and enhanced cytoplasmic granularity. Increased CD14 expression was seen in 4 of the 6 experiments (median, range % increase: 11.2%, 2.5%-21%), consistent with myeloid plasticity; Increased KLRG1 expression was seen in 5 of the 7 experiments (median MFI ratio, range: 3.7, 1.3-471). No significant change was seen in GPNMB expression. Compared to H929VEH, H929HDM had a reduction in killing by CAR-T cells (median±SD: 22.8±32.94% n=6, p=0.016, Wilcoxon paired test). Similarly, H929HDM had a 10.69% decrease in Tec-mediated T-cell killing compared to H929VEH (n=8, p=0.02, Wilcoxon paired test). Conclusions: Late-stage MM cells exhibit transcriptional signatures consistent with TIS, which can be associated with decreased PFS to CAR-T. We report an in vitro model of TIS MM cells for testing of resistance to T cell killing. Based on these findings, a phase II trial (NCT06940297) is underway to test the hypothesis that peri-CAR-T senolytic therapy with dasatinib and quercetin will deepen responses by targeting this resistant TIS MM population. Citation Format: Christoph Schaefers, Angelo Jose Guilatco, Sofia Arbelaez, Gabriel Alvares Borges, Chen Wu, Andre de Menezes Silva Corraes, Malvika Gupta, Zuoyi Shao, Kevin Regan, Rayaan Kamal, Neal I. Sannuli, Ying Li, Taxiarchis Kourelis, Wilson Gonsalves, Melinda Tan, Nadine Abdallah, Yi Lin, Megan Weivoda. Myeloma cells with therapy-induced senescence-like phenotype have increased resistance to killing by T cell directed therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6475.

Abstract Estrogen receptor alpha (ERα)-positive breast cancer accounts for up to 80% of cases. Despite advances in CDK4/6 inhibitors (CDK4/6i) and endocrine therapies, recurrence and late relapse remain significant challenges. Recurrence often arises from disseminated cancer cells that persist despite 5-10 years of systemic adjuvant treatment. While CDK4/6i and endocrine therapies induce prolonged growth arrest resembling senescence, they do not significantly kill cancer cells. Senescent/pseudo-senescent cancer cells are implicated in aggressive variant formation, recurrence, and metastasis. We explored eliminating breast cancer cells via senolysis while they are in therapy-induced senescent state to prevent their potential to escape, repopulate, or cause long-term adverse effects. Live-cell imaging of MCF7 and T47D cells treated with abemaciclib and endocrine therapies (endoxifen or estrogen deprivation to mimic aromatase inhibition) at clinically relevant concentrations for eight days demonstrated prolonged growth cessation and G1 cell cycle arrest even two weeks post-drug withdrawal. These cells displayed a senescence-like gene expression profile, with increased CDKN2B (p15), CDKN1A (p21), BCL2, reduced KI67, and increased TGFβ expression, a known Senescence-Associated Secretory Phenotype (SASP) factor linked to deep senescence. These results confirm effective senescence induction by various CDK4/6i and endocrine therapy combinations. Further analysis using established senescence markers (e.g., nuclear size, telomere-associated foci, satellite distension, and loss of nuclear HMGB1 and LaminB1) revealed unique senescence phenotypes across therapy combinations. Cell viability assays and live imaging of Caspase 3/7 activation demonstrated differential vulnerabilities of senescent/senescent-like ERα+ cancer cells (MCF7, T47D, UCD4, UCD12) to 13 senolytics (1 μM), including natural flavonoids and inhibitors targeting pro-survival factors, tyrosine kinases, and the Notch pathway. For example, MCF7 and UCD4 cells responded to MCL1i and RSL3, T47D to BCLXLi and RSL3, and UCD12 to BCLXLi and Navitoclax. Notably, senolytic cocktails (e.g., RSL3, GNF7, Venetoclax, Dasatinib) at reduced concentrations (0.25 μM each) effectively eliminated senescent/senescent-like ERα+ cancer cells across all cancer cell lines, achieving 100% elimination in T47D cells. Collectively, these findings demonstrate that despite variability in therapy-induced senescence, senolytic combinations effectively target senescent ERα+ cancer cells. This provides preliminary evidence for the potential integration of this strategy into standard breast cancer therapy with a goal to improve recurrence-free survival and increase the cure rates for ERα+ breast cancer patients. In vivo studies are underway to evaluate this strategy in preclinical models. Citation Format: Lena Batoon, Angelo J. Guilatco, Anna Detry, Xiyin Wang, Megan M. Weivoda, John R. Hawse. CDK4/6 inhibitors and endocrine therapies induce senescence with drug- and cell line-specific vulnerabilities in ERα+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 519.

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. Current standard of care includes autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) administered as a conditioning agent. Cytotoxic chemotherapies such as melphalan have been shown to drive therapy-induced senescence (TIS) in several tissues. We hypothesize that HDM activates TIS pathways in surviving MM cells and that this may correlate with patient outcomes post-ASCT. We developed an in vitro HDM model using 5TGM1 mouse MM cells treated with vehicle (Veh) or HDM (10µM, IC90) for 6h, followed by co-culture with primary mouse bone marrow stromal cells. Image analysis was performed to quantify proliferation by cumulative population doublings (CPDs) over 10 days, after which 5TGM1 cells were evaluated for cell size, telomere-associated DNA damage foci (TAFs), sensitivity to senolysis, and senescence gene expression (scRNA-seq and RT-qPCR). The effect of HDM on CPDs and cell size was also evaluated in human MM cell lines RPMI-8226 and H929. scRNA-seq was used to analyze CD138+ PCs from MM patients at various timepoints post-ASCT. Longitudinal MM patient iliac crest biopsies (diagnosis, post-ASCT; N=20) were stained for CD138+ PCs; PC burden was quantified by AI-assisted histology. Lastly, we established an in vivo model of induction therapy (bortezomib) followed by HDM/SCT in Vk*MYC mouse MM engrafted C57BL/6 mice. Tumor burden was tracked by serum protein electrophoresis for M-spike using submandibular bleeds. Bone marrow was then flushed from long bones and analyzed by flow cytometry and immunofluorescence. HDM-5TGM1 showed decreased CPDs (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001, N=3), increased size (median pixel size: HDM 413.2±99.98, Veh 207±8.88, p<0.05, N=3), and increased TAF+ cells (Percent TAF+ cells: HDM 34.77%±10.74%, Veh 11.41%±1.58%, p<0.05, N=3) consistent with known TIS features. Preliminary assessment (N=1) of human MM cell lines showed similar growth arrest (RPMI-8226 CPDs: HDM 0.058±0.101, Veh 4.185±0.358; H929 CPDs: HDM 0±0, Veh 4.61±0.243) and size increase (RPMI-8226 mean pixel size: HDM 804.1±6.048, Veh 526.9±2.115; H929 mean pixel size: HDM 371.2±26.95, Veh 130.3±41.43), suggesting a translatable mechanism. HDM-5TGM1 cells were more sensitive to senolysis by dasatinib+quercetin (viability: HDM 54.9%±0.09%, Veh 76.0%±0.05%, N=2). scRNA-seq analysis showed that HDM-5TGM1 were enriched for gene sets related to senescence pathways, including the published set SenMayo (FDR<0.25). HDM-5TGM1 cells also exhibited myeloid gene expression consistent with previous publications on MM dormancy. RT-qPCR confirmed significant increases in HDM-5TGM1 (p<0.001, N=3) in senescence markers (Cdkn1a, Cdkn1c, Glb1), senescent cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), senescence associated secretory phenotype (SASP) genes (Ccl5, Icam1, Mmp13), and myeloid markers (Axl, Fcer1g, Mpeg1). scRNA-seq of patient CD138+ PCs confirmed a myeloid-like cluster that gained prominence in relapsed patients with resistant disease. CD138+ PCs were detected in patient bone biopsies post-ASCT. Interestingly, patients who relapsed (≤3 years, N=9) had a greater percent reduction in PC burden from diagnosis to post-ASCT when compared to patients that did not relapse (N=11) in that time (p=0.02). Further, CD138+ PC burden post-ASCT positively correlated with time in remission in relapsed patients (R=0.697, p=0.03). Bortezomib reduced serum M-spike in Vk*MYC-engrafted mice (p=0.0003); M-spike was further reduced by HDM/SCT compared to Veh-treated mice (p=0.0007). Flow cytometry confirmed a decrease in Vk*MYC cells after HDM/SCT (p=0.0043), and the percentage of Vk*MYC cells with greater size and complexity was increased compared to Veh-treated mice (p=0.0043), consistent with our in vitro data. Vk*MYC cells from HDM-treated mice also trended for more TAF+ cells (>3 TAFs) compared to Veh-treated mice (p=0.0714). Our findings suggest that HDM can induce a TIS and myeloid signature in surviving MM cells. Greater MM burden post-ASCT appears to be associated with longer durable response, again suggesting that these cells may be in growth arrest consistent with TIS. Finally, senolytics may be a novel approach to eliminate surviving MM cells and prevent relapse.

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2958.

Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. One mechanism that may contribute to MM relapse is tumor dormancy. Dormant MM cells may persist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells may then exit dormancy following treatment to drive disease relapse. Dormancy shares many features with cellular senescence, a state of stable growth arrest induced by cell stress. Chemotherapies that produce genotoxic stress have been associated with therapy-induced senescence (TIS). One such genotoxic agent, melphalan (Mel), is routinely administered to MM patients for myeloablation prior to autologous bone marrow transplant. We hypothesized that High Dose Mel (HDM) induces a senescent-like dormant phenotype in surviving MM cells. To test our hypothesis, we developed an in vitro model for HDM. We identified 10µM Mel as a concentration to induce >90% cell toxicity (HDM) in 5TGM1 mouse MM cells over 10 days. 5TGM1-GFP MM cells were cultured with HDM or vehicle (Veh) for 6 hours; cells were then cultured in normal growth medium in the presence of primary mouse (C57BL/6) bone marrow stromal cells (BMSCs). Co-cultures were imaged by fluorescent microscopy every other day to quantify cumulative population doublings (CPD) and cell morphology over the following 10 days. The effect of HDM on CPDs was also evaluated in the human RPMI-8226 MM cell line. 5TGM1-GFP cells were isolated by FACS on day 10 for evaluation of senescence histological characteristics and gene expression. All studies represent n=3 independent experiments unless otherwise indicated. HDM-5TGM1 cells exhibited a significant reduction in mean CPDs over 10 days compared to Veh (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001), indicating stable growth arrest in HDM treated cells consistent with senescence. HDM-5TGM1 cells were also significantly larger versus Veh (median size in pixels units: HDM 413.2±99.98, Veh 207±8.88, p<0.05), consistent with classical enlarged morphology in senescent cells. Preliminary assessment (n=1) of HDM-RPMI-8226 cells showed similar growth arrest (mean cumulative doublings: HDM 0.058±0.101, Veh 4.185±0.358) and enlargement (mean size in pixel units: HDM 804.1±6.048, Veh 526.9±2.115), suggesting a translatable mechanism in humans. FACS-sorted 5TGM1-GFP cells were stained for telomere associated foci (TAF), a marker of persistent DNA damage characteristic of senescent cells. HDM-5TGM1 cells showed a higher percentage of TAF+ cells (Percent TAF+ cells: HDM 39.7%±9.1%, Veh 10.6%±0.8%, n=2). Sorted HDM-5TGM1 cells also exhibited significant increases over Veh (p<0.001) in senescence markers ( Cdkn1a, Cdkn1c, Glb1), senescence cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), and senescence associated secretory phenotype (SASP) genes ( Ccl5, Icam1, Mmp13). In addition, HDM-5TGM1 cells showed significant increases in myeloid markers that have previously been shown to be increased in dormant 5TGM1 ( Axl, Fcer1g, Mpeg1). Single cell RNA-sequencing was used to further evaluate senescence gene expression changes in growth arrested HDM-5TGM1 cells. Gene set enrichment analysis (GSEA) of genes differentially expressed in HDM-5TGM1 versus Veh cells was performed for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescence Growth Arrest, SenGA; Inflammatory SASP, iSASP; SCAPs; and Plasma Cell Senescence; PCSen) as well as mouse ortholog versions of a published senescence and biological aging gene set (SenMayo) and KEGG curated gene sets. GSEA of HDM-5TGM1 versus Veh cells confirmed significant (FDR<0.25) upregulation of iSASP, MayoSen, PCSen, SenUp, SCAPs, and SenGA gene sets. Concurrently, GSEA revealed significant downregulation of KEGG DNA replication, cell cycle, and nucleotide excision repair gene sets, supporting our observations of proliferative arrest and persistent DNA damage TAF. Our findings demonstrate that HDM-MM cells exhibit multiple features of senescence, suggesting a potential mechanistic role for TIS in MM dormancy and highlight a novel therapeutic target that may be present in patients post-HDM. Thus, targeting senescence may be a novel approach to eliminate dormant MM cells and prevent relapse.

Multiple Myeloma (MM) progresses from monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Despite similar primary genetic events as MM, MGUS and SMM PCs are non- or low-proliferative. Since oncogene activation and DNA damage drive senescence growth arrest to promote immune clearance of potentially malignant cells, we hypothesized that MGUS and SMM pre-malignant PCs exhibit senescence features. To test this, we performed gene set enrichment analysis (GSEA) of a published human PC gene array dataset (GSE5900) comparing normal, MGUS, and SMM PCs for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescent Cell Anti-Apoptosis Pathways, SCAPs; Senescence Growth Arrest, SenGA; Inflammatory Senescence Associated Secretory Phenotype, iSASP; Interferon SASP, IFN-SASP) versus biological aging gene sets (SenMayo; CellAge). MGUS and SMM PCs exhibited significant enrichment (NES>1.5, q<0.05) for senescence gene sets SenUp, SenGA, and SCAPs compared to normal PCs, with no enrichment for aging gene sets. MGUS PCs were also enriched for iSASP (NES=1.4, q=0.11), whereas SMM PCs were enriched for IFN-SASP (NES=2.3, q<0.05), characteristic of late-senescence. Thus, both MGUS and SMM PCs show enrichment of senescence gene sets compared to normal PCs with differential SASP profiles. Next, we evaluated MGUS, SMM, and MM patient PCs isolated by magnetic sorting for histological senescence features. PCs were immunostained for LMNB1 and HMGB1, and fluorescent in situ hybridized for α-satellite. Cellular senescence was defined as loss of ≥50% nuclear membrane LMNB1, loss of HMGB1, and ≥2 senescence-associated distensions of satellite (SADS) per nuclei. PCs from MGUS patients (N=12) exhibited increased percentage of senescent PCs (26.9±4.1%, Mean±SEM, p<0.0001) compared to SMM (11.0±1.9%, N=19, p<0.0001) and MM (11.6±1.5%, N=11, p<0.001). A subset analysis of SMM patients for which disease stability was known (≥2 years, N=6-8) showed no differences. These results demonstrate that the percentage of senescent PCs is increased in MGUS but not SMM PCs compared to proliferative disease. To assess PC senescence in the native bone marrow microenvironment (BMME), we evaluated trephine bone biopsies from MGUS and SMM patients that progressed or not to MM ≤10 years, as well as newly diagnosed MM. Biopsies were immunostained for CD138, LMNB1, and HMGB1. Whole biopsy images were analyzed using the artificial intelligence-based software HALO. Cells were classified as senescent if they were double negative and non-senescent if they were double positive for LMNB1 and HMGB1. The percentage of non-senescent PCs increased with progression to MM and positively correlated with total PC burden (R=0.52, p<0.0001), reflective of proliferative disease. Senescent PCs correlated with total senescent BMME (R=0.87, p<0.0001), consistent with a role for senescent cells to drive paracrine senescence. We performed proximity analysis to evaluate the cellular composition within 25um of senescent and non-senescent PCs. As expected, non-senescent PCs exhibited a significantly greater percentage of proximate non-senescent BMME in all groups (p<0.0001). In contrast, senescent PCs in stable MGUS (N=16) and SMM (N=14) were equally surrounded by senescent and non-senescent BMME. In progressing MGUS (N=21) and SMM (N=20), as well as MM (N=18), senescent PCs exhibited significantly reduced senescent BMME in their proximate environment (p<0.05). This suggests that senescent PCs in progressive disease, in contrast to stable disease, fail to drive local paracrine senescence responses. These data demonstrate that PCs from stable MGUS and SMM patients exhibit senescence features, with isolated PCs from stable MGUS showing increased senescence gene expression and histologic features. While stable SMM also exhibits senescence gene expression, they lose morphological features of senescence and show a preferential IFN SASP. Within the bone compartment, senescent PCs in stable MGUS and SMM exhibit the expected proximity to senescent BMME. In contrast, progressing MGUS and SMM, as well as NDMM, show a loss of proximate senescent BMME. Given the role of senescence to drive inflammation to clear potentially tumorigenic cells, these data support that failure to drive paracrine senescence may be key to the immune escape of pre-malignant cells in MGUS and SMM.

Abstract Multiple myeloma (MM) is a clonal plasma cell (PC) cancer that is preceded by the benign conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). While MGUS/SMM PCs are not proliferative, many of the oncogenes and chromosomal abnormalities in MM PCs are also present in MGUS/SMM. Since oncogenic stress is known to induce cellular senescence, we hypothesized that MGUS/SMM PCs may be in a senescent-like state. Our analysis of a published human dataset (GSE5900) revealed that compared to healthy PCs, MGUS/SMM PCs had significantly increased expression of senescence markers CDKN1A and GADD45A (FDR<0.05). Gene Set Enrichment Analysis (GSEA) showed significant enrichment in MGUS/SMM PCs of our customized senescence phenotyping gene sets (q<0.25). We next evaluated the KaLwRij MGUS mouse model. 10-mo-old female KaLwRij mice were treated with placebo or senolytics (dasatinib+quercetin, D+Q). D+Q treatment significantly reduced PCs compared to placebo, while restoring B cell number and functional gene expression. PCs isolated from D+Q-treated KaLwRij mice exhibited significant reductions in Myc and Il1b expression (p<0.05) versus placebo and a trend towards reduced Trp53 expression (p=0.13), supporting that senescent PCs are targeted by D+Q. Single-cell RNA-seq and single sample (ss)GSEA of 24-mo-old KaLwRij PCs revealed a PC subset enriched for the ‘Plasma Cell Senescence’ gene set, which included genes from our customized gene sets that were differentially expressed in the human PCs dataset GSE5900. Notably, PCs in this subset showed enrichment for ‘Inflammatory SASP’ (median: 15.26%) and ‘Interferon (IFN) SASP’ (39.36%) gene sets. The IFN-SASP is characteristic of late senescence, driven by the accumulation of transposable elements. In the GSE5900 dataset, we found significant enrichment of ‘IFN-SASP’ in SMM (q<0.25) but not MGUS PCs. Further, whole transcriptome RNA-sequencing showed that SMM PCs had increased expression of LINE1 retrotransposon L1HS in relation to normal PCs (FDR<0.1), but not MGUS (FDR=0.83) or MM (FDR=0.5) PCs. Immunostaining confirmed increased cytosolic DNA:RNA hybrids in SMM (median frequency of cells exceeding the intensity threshold = 33%) versus MGUS (16.4%), MM (4.4%), and healthy PCs (1.5%), which is consistent with cytosolic DNA-mediated activation of the IFN SASP in SMM PCs. When these same patients were redistributed according to disease progression, L1HS was higher in patients with progressing SMM and newly diagnosed MM (NDMM) (FDR<0.1) versus patients with stable MGUS (FDR=0.56) or advanced MM (FDR=0.38). Increased DNA:RNA staining was observed in progressing SMM PCs (median frequency = 41%), but the results for NDMM patients (4.4%) and advanced MM (3%) were comparable to healthy or stable patients (3.5%). These data demonstrate that MGUS and SMM PCs exhibit senescence features, suggest mechanisms that may contribute to MM tumorigenesis, and show that pharmacological ablation of senescent cells may prevent progression from MGUS/SMM to MM. Citation Format: Gabriel Alvares Borges, Angelo Jose Guilatco, Christine M. Hachfeld, Ming Ruan, Sonya Royzenblat, Ming Xu, Claire M. Edwards, Marta Diaz-delCastillo, Thomas L. Andersen, Taxiarchis Kourelis, Tamar Tchkonia, James L. Kirkland, Matthew T. Drake, Megan Weivoda. Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR012.

Abstract Multiple myeloma (MM) is an incurable plasma cell cancer. Therapeutic advances have greatly improved patient survival; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive relapse. Tumor dormancy shares features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the treatment of MM, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that depends on interactions with the mesenchymal bone niche. GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of senescence after the initial stress response. Cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and stained for nuclei, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly growth arrested over 2 weeks compared to vehicle-treated cells. Co-culture with BMSCs significantly enhanced melphalan-treated 5TGM1 adherence compared to culture without BMSCs during media replacement. Of note, melphalan-treated 5TGM1 did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 also exhibited significantly greater adherence with BMSCs than vehicle-treated cells. Melphalan-treated 5TGM1 were significantly larger than vehicle-treated cells and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, which are all markers of cellular senescence. Our findings suggest that interactions with BMSCs promote the survival of melphalan-treated MM tumor cells. The enhanced adherence of these cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse. Citation Format: Angelo J. Guilatco, Gabriel Alvares Borges, Tamar Tchkonia, James L. Kirkland, Taxiarchis Kourelis, Matthew T. Drake, Megan Weivoda. Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B023.

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR012) of the Conference Proceedings. Citation Format: Gabriel Alvares Borges, Angelo Jose Guilatco, Christine M. Hachfeld, Ming Ruan, Sonya Royzenblat, Ming Xu, Claire M. Edwards, Marta Diaz-delCastillo, Thomas L. Andersen, Taxiarchis Kourelis, Tamar Tchkonia, James L. Kirkland, Matthew T. Drake, Megan Weivoda. Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B020.