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Salivary glands are common organs at risk in both head and neck external beam radiotherapy (EBRT) and radiopharmaceutical therapy (RPT), but incidences of xerostomia in RPT are inconsistent with the EBRT Quantitative Analysis of Normal Tissue Effects in the Clinic (QUANTEC) limits. In EBRT, salivary glands are usually assumed to be parallel organs, with QUANTEC guidelines based on Dmean, but this is known to be a gross over-simplification of the full complexity of the underlying functional organization. The goal of this work is to combine machine learning of EBRT dose–outcome data with stylized small-scale RPT dosimetry to discover more reliable normal tissue complication probability (NTCP) models of xerostomia across both modalities. A retrospective cohort of 211 EBRT patients was analyzed using a custom-designed in-house machine learning workflow. From this, a hierarchy of three models of increasing complexity was trained, evaluated for performance and generalization, and coupled with stylized small-scale salivary gland dosimetry to assess the influence of model complexity on the predicted NTCP for plausible patterns of RPT dose nonuniformity. The three models in the hierarchy (A, B, C), in increasing order of complexity, associate xerostomia with the following: the mean dose to the whole contralateral parotid (model A), the mean dose to a ductally localized region (model B) and a serial interaction dose term between two ductal sub-compartments (model C). While the difference between the three models for EBRT p-values and AUCs is rather marginal, for physiologically driven ductal dose distributions in RPT, the predicted reduction in TD50 can be as large as a factor of 10. These results provide hints towards a plausible reconciliation of the observed inconsistency of xerostomia in RPT with EBRT dose limits.

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription factors (MTFs) GATA6 and ΔNp63, respectively. Objective This study sought to uncover the transcriptional regulatory mechanisms controlling PDAC subtype identity. Design We integrated primary tumour single-cell RNA-seq, patient-derived xenograft RNA-seq and multispectral imaging to identify MTF-dependent, subtype-specific markers. We created subtype-specific fluorescent reporter systems and conducted drug screenings to find actionable targets. We analysed chromatin accessibility (ATAC-seq), genome-wide occupancy (ChIP-seq) for epigenetic status (H3K27ac), MTFs (GATA6, ΔNp63), RNA polymerase II (Pol II), H3K4me3-anchored chromatin topology (HiChIP) and nascent RNA capture sequencing (PRO-seq). Additionally, we used nuclease-dead Cas9 (dCas9) to manipulate transcriptional regulatory mechanisms. Results Our approach identified glucocorticoid receptor (GR) agonists as agents that suppress the classical transcriptional programme by interacting with GATA6. GATA6 regulates classical-specific transcription through promoter-proximal pause release. Depletion of GATA6 increased Pol II occupancy at GATA6-bound enhancers and transcriptional start sites, stabilising enhancer–promoter interactions. Artificially inducing pausing at GATA6-bound enhancers with dCas9 abrogated target gene expression and induced pausing at both the enhancer and target gene promoter. Conversely, in basal PDAC ΔNp63 promotes Pol II recruitment and stabilises enhancer–promoter interactions. Conclusion This study provides new insights into the transcriptional control and role of GR agonists in controlling PDAC molecular subtype identity.

Introduction: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral small vessel disease, yet there is currently no biomarker for early detection. This study aims to identify retinal biomarkers of NOTCH3 carriers using optical coherence tomography (OCT) from the UK Biobank. Methods: We conducted a cross-sectional, matched case-control study of individuals with or without CADASIL from the UK Biobank between 2006 and 2010. All participants had retinal OCT scan and cognitive assessment. Cases were identified based on pathogenic NOTCH3 mutations and 1:1 matched with healthy controls based on age, sex, hypertension, diabetes mellitus, and smoking status. Baseline characteristics and cognitive test scores were compared using chi-square or Mann-Whitney-U test appropriately. Macular thickness (central, inner, and outer subfields) (Figure) and retinal nerve fiber layer (RNFL) thickness were compared using Wilcoxon signed-rank test. Results: Our analysis included 134 participants (67 CADASIL cases, 67 controls) with a mean age of 54±8 years. There were no differences in visual acuity between the two groups. CADASIL performed worse in prospective memory (0.043), verbal and numerical reasoning (p=0.178), visual memory (p=0.227), and processing speed (p=0.101) than matched controls. Participants with CADASIL had significantly thinner macular inner subfield at the superior (p=0.006), temporal (p=0.008), and nasal (p=0.034) quadrants, and significantly thinner RNFL (p=0.008) compared to controls (Table). Conclusion: Individuals with CADASIL have reduced thickness in the inner macular subfield and RFNL relative to matched controls. These retinal group differences were not associated with differences in visual acuity and may reflect early pericyte dysfunction and microvascular ischemia. Longitudinal studies are needed to assess the temporal relationship between these retinal changes and disease progression.

Abstract Recent clinical trials on slowing dementia progression have led to renewed focus on finding safer, more effective treatments. One approach to identify plausible candidates is to assess whether existing medications for other conditions may affect dementia risk. We conducted a systematic review to identify studies adopting a data‐driven approach to investigate the association between a wide range of prescribed medications and dementia risk. We included 14 studies using administrative or medical records data for more than 130 million individuals and 1 million dementia cases. Despite inconsistencies in identifying specific drugs that may modify Alzheimer's or dementia risk, some themes emerged for drug classes with biological plausibility. Antimicrobials, vaccinations, and anti‐inflammatories were associated with reduced risk, while diabetes drugs, vitamins and supplements, and antipsychotics were associated with increased risk. We found conflicting evidence for antihypertensives and antidepressants. Drug repurposing for use in dementia is an urgent priority. Our findings offer a basis for prioritizing candidates and exploring underlying mechanisms. Highlights We present a systematic review of studies reporting association between drugs prescribed for other conditions and risk of dementia including 139 million people and 1 million cases of dementia. Our work supports some previously reported associations, for example, showing decreased risk of dementia with drugs to treat inflammatory disease and increased risk with antipsychotic treatment. Antimicrobial treatment was perhaps more surprisingly associated with decreased risk, supportive of recent increased interest in this potential therapeutic avenue. Our work should help prioritize drugs for entry into adaptive platform trials in Alzheimer's disease and will serve as a useful resource for those investigating drugs or classes of drugs and risk of dementia.

Background: Very-late-onset myasthenia gravis (VLOMG) refers to myasthenia gravis (MG) with onset at age 65 or older. Current research on VLOMG prognosis remains limited, especially regarding factors influencing outcomes. Objectives: To identify the clinical factors that affect the short- and long-term prognosis of MG patients with an onset age ⩾65 years. Design: This was a single-center, retrospective cohort study of AChR-ab positive VLOMG patients, classified into two subgroups based on age of onset: sub-very-late-onset MG (S-VLOMG, onset age ⩾65 and <75 years), and super-late-onset MG (SLOMG, onset age ⩾75 years). Methods: A total of 93 patients were included, including 75 in the S-VLOMG group and 18 in the SLOMG group. Clinical, therapeutic, and prognosis data were reviewed, and the Cox regression model was used to identify factors influencing short- and long-term prognosis. Results: Patient characteristics were well balanced between the groups. Overall, 49.5% of patients achieved minimal symptom expression (MSE) within 6 months and 86% within 24 months. There was no significant difference between the groups in the proportion achieving MSE at 6 months ( p = 0.635) or 24 months ( p = 0.714). The median time to achieve MSE was also comparable between the S-VLOMG and SLOMG groups (199.0 days vs 280.5 days, p = 0.463). Low baseline MG-ADL score and steroid therapy were associated with better short-term prognosis ( p = 0.007 and p = 0.002, respectively). For long-term prognosis, baseline bulbar and limb involvement, time to treatment initiation, and use of immunosuppressants were significant factors ( p = 0.025, p = 0.004, p = 0.025, and p < 0.0001, respectively). There were no significant differences in side effects or drug withdrawal rates between two groups. Conclusion: This study demonstrated that AChR-ab positive VLOMG patients have a favorable prognosis and responded well to medication, with age and comorbidities showing no significant impact.

Abstract Introduction Incorporation of mammographic density to breast cancer risk models could improve risk stratification to tailor screening and prevention strategies according to risk. Robust evaluation of the value of adding mammographic density to models with comprehensive information on questionnaire-based risk factors and polygenic risk score is needed to determine its effectiveness in improving risk stratification of such models. Methods We used the Individualized Coherent Absolute Risk Estimator (iCARE) tool for risk model building and validation to incorporate density to a previously validated literature-based model with questionnaire-based risk factors and a 313-variant polygenic risk score (PRS). The model was evaluated for calibration and discrimination in three prospective cohorts of European-ancestry women (1,468 cases, 19,104 controls): US-based Nurses’ Health Study (NHS I and II) and Mayo Mammography Health Study (MMHS); and Sweden-based Karolinska Mammography Project for Risk Prediction of Breast Cancer (KARMA) study. Analyses were done separately for women younger (NHS II, KARMA) and older than 50 years (NHS I, MMHS, KARMA). Improvements in terms of risk stratification and reclassification proportions were assessed among European-ancestry women aged 50–70 years in US and Sweden. Results For women younger and older than 50 years, the model with questionnaire-based risk factors, PRS and density was generally well calibrated across risk with some evidence of miscalibration at the extremes of the risk distribution. Incorporation of density led to modest improvements risk discrimination beyond the model with questionnaire-based risk factors and PRS: the area under the curve (AUC) among younger women was 67.0% (95% CI: 63.5–70.6%) vs. 65.6% (95% CI: 61.9–69.3%) for models with and without density; and 66.1% (95% CI 64.4–67.8%) vs. 65.5% (95% CI: 63.8–67.2%) among older women. The model with density identified 18.4% of US women 50–70 years old ≥ 3% 5-year predicted risk (threshold used for recommending risk-reducing medication in the US), with 42.4% of future cases expected to occur in this group. At this threshold, 7.9% of US women were reclassified by adding density to the model, resulting in the identification of 2.8% of additional future cases. The model with density identified 10.3% of Swedish women ≥ 3% 5-year predicted risk, with 29.4% of future cases expected to occur in this group. At this threshold, 5.3% of women were reclassified with the addition of density, leading to the identification of an additional 4.4% of future cases. Conclusion Integrating density with questionnaire-based risk factors and PRS could potentially identify more women of European-ancestry with elevated risk of breast cancer in the United States and Sweden. Further investigations of the integrated model in non-European ancestry populations are needed prior to considering clinical applications.

Overview: Despite advances in therapies targeting the tumor-intrinsic aspects of the disease, multiple myeloma (MM) remains functionally incurable. Given that new alternatives such as T cell redirected therapy and monoclonal antibodies engage ADCC and CDC by leveraging other elements of the immune system, we believe that deeper studies of the tumor microenvironment are warranted. In particular, we believe that certain macrophages are associated with tumor progression, serve as markers of future relapses, are immunosuppressive, and reduce the effectiveness of otherwise successful therapies. Methods: We performed 5' scRNAseq analysis of 189,025 cells collected from spleen or bone marrow of 4 wild type, 14 de novo, and 40 tumor bearing Vk*MYC mice treated with vehicle or effective anti-MM therapies. Discoveries were cross-referenced against a set of 45,626 human bone marrow cells collected from 35 samples at different stages of disease and treatment. We experimentally recreated in vitro the cross-talk between MM cells and macrophages by co-culturing bone marrow derived macrophages (BMDM) with MM cells or their conditioned medium, and correlated changes in gene expression as well as the ability of polarized BMDM to inhibit CAR T mediated killing of MM cells in vitro. Results: Gene expression analysis of murine in vivo C1q macrophages and in vitro polarized BMDM revealed a common signature identified in lipid-associated macrophages and Kupffer cell-like macrophages1 (high expression of Fabp5, ApoE, Cd63, C1qa/b/c, Cd163, Cd5l, Vcam1, Fcgr4, Folr2, Maf) which is associated with immunosuppression and disease progression. Ligand-receptor analysis via LIANA identified C1qb ligand in macrophages with C1qbp in tumor among the top interactions in both human and murine single cell analyses. C1qbp is a crucial element of cell adhesion and metastasis,2 and this link between tumor and macrophage can severely limit effective therapy: BMDM following stimulation/polarization with MM lines, but not naïve/unstimulated BMDM, strongly inhibit anti-BCMA CAR T in vitro cytotoxic activity. In total, a complement-related connection between tumor and macrophage is capable of directing an immunosuppressive, chemokine response that imparts resistance to therapy. While effective therapies reduce tumor and C1q expressing macrophage populations,3 retention of C1q expressing cells may be associated with future relapses. Presence of a specific subset of C1Q expressing cells, even in very low numbers, in human samples following ASCT was associated with early relapse (6 of 8 good responding samples had zero C1Q+ cells while 12 of 16 poor responding samples had 1 or more C1Q+ cells-Fisher test p-value &lt; 0.05). In addition to high expression of C1QA/B/C, these cells are also highly upregulated in TYROBP (known to relate to macrophage polarization4 and found in a spatial transcriptomic analysis of cells at the periphery of MM and its environment5) as well as FCER1G (known to relate to macrophage infiltration6). Conclusion: As advanced therapies manipulate elements of the immune microenvironment to impart effective treatment, the connection between tumor and macrophage should not be overlooked since macrophages likely help create an immunosuppressive niche that assists in disease growth and eventual revival. 1. Ma, R.-Y., Black, A. & Qian, B.-Z. Macrophage diversity in cancer revisited in the era of single-cell omics. Trends Immunol. 43, 546-563 (2022). 2. Wang, Y. et al. C1QBP suppresses cell adhesion and metastasis of renal carcinoma cells. Sci. Rep. 7, 999 (2017). 3. Stein, C. K., Meermeier, E., Welsh, S. J., Chesi, M. & Bergsagel, P. L. Key Subsets of Immune Cells in Bone Marrow Myeloid Compartment Indicate Degree of Tumor Infiltration and Avenues for Successful Immunotherapy in Multiple Myeloma. Blood 140, 7082-7083 (2022). 4. Liang, T. et al. TYROBP, TLR4 and ITGAM regulated macrophages polarization and immune checkpoints expression in osteosarcoma. Sci. Rep. 11, 19315 (2021). 5. Sudupe, L. et al. Spatial Transcriptomics Unveils Novel Potential Mechanisms of Disease in a MI cγ1 Multiple Myeloma in vivo Model. Blood 142, 4076 (2023). 6. Dong, K. et al. FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity. BMC Cancer 22, 140 (2022).

Multiple myeloma (MM), an incurable hematologic malignancy, presents significant challenges in managing its recurrence. The bone marrow tumor microenvironment (BM-TME) is critical to MM progression. However, classical immune checkpoint blockade (ICB) therapies, like anti-PD1, have shown limited efficacy in MM. Therefore, elucidating the compositional features and regulatory mechanisms of the TIME in MM is crucial for developing novel immunotherapeutic targets. Tissue-resident memory CD8+ T cells (CD8+ TRMs) reside indefinitely at multiple anatomical locations like the skin, intestine, and bone marrow, functioning as frontline responders against reencountered pathogens and tumors. Whether CD8+ TRMs within BM-TME are involved in the MM cell immune escape and pathogenesis has not been systematically elucidated. Firstly, we leverage single-cell RNA sequencing (scRNA-seq) to dissect the BM CD8+ T cells across patients at different stages of MM, and identify that CD69+CD8+ T subsets were BM residents, and the proportion of BM CD8+ TRMs between health donors and MM patients was comparable. Moreover, our findings indicated that BM CD8+ TRMs exhibit exhausted features and enrichment of NK genes with MM progresses. Importantly, we observed a considerable elevation of the NK gene KLRB1 and its encoded protein CD161 in BM CD8+ TRMs in MM patients. Immunofluorescence labeling of BM biopsy samples from MM patients confirmed the infiltration of the BM-TME with CD3+CD8+CD69+CD161+ cells. Then, we found that KLRB1 expression in BM CD8+ TRMs was higher than that of other classical inhibitory receptor genes (e.g., PDCD1, CTLA4, LAG3), and CLEC2D (encodes the ligand of CD161) was also markedly expressed in patients' MM cells, exhibiting considerably higher than classical immunosuppressive receptor ligand genes (e.g., PDL1, CD80, CD86). Moreover, patients with higher CLEC2D expression had unfavorable overall survival. We further found that the CD161 expression was higher in CD69+CD8+ TRMs compared to circulating CD69-CD8+ T cells, and the percentage of BM CD161+CD8+ TRMs in MM patients was significantly higher than in healthy donors, while there was no difference for PD-1, LAG3, and TIM3 expression. Notably, only a small fraction of BM CD8+ TRMs have mucosal-associated invariant T cell phenotypes (identified by CD161+TCR-Vα7.2+). Additionally, the CLEC2D expression of BM cells was analyzed, and the highest level of CLEC2D expression was observed in B cells from MM patients, followed by plasma cells. Importantly, CLEC2D expression in MM patients was significantly higher than in plasma cells from healthy donors. Next, to investigate whether the blockade of CD161 could restore the function of CD8+ TRMs, the primary BM cells from MM patients were isolated and stimulated ex vivo. Compared to the anti-PD-1 and isotype group, CD161 blocking presents enhanced CD8+ TRMs proliferation and cytokine secretion levels (TNF-α, IFN-γ, IL2), increasing MM cells apoptosis. Chimeric antigen receptor T (CAR-T) cell therapy has achieved impressive success in treating relapsed/refractory MM. How to prevent CAR-T cell exhaustion and enhance function remains a significant challenge. Therefore, we next set out to investigate the impact of CD161 on CAR-T cells. Our results demonstrated that CD161 was highly expressed in early peripheral blood (PB) CAR-T cells after infusion in MM patients but decreased with time. Moreover, the CD161 expression of CAR-T cells was considerably higher than endogenous T cells. Notably, BM-derived CAR-T cells have higher CD161 expression than PB counterparts for the same patient, suggesting CD161 may have BM-specific expression enrichment. After co-culturing MM and CAR-T cells in vitro for 24 hours, we found that blocking CD161 led to an augmented proportion of CAR-T cells, increased IFN-γ secretion, and a notable elevation in tumor cell apoptosis compared to the anti-PD-1 and isotype groups. Taken together, we demonstrated that CD8+ TRMs constitute a vital anti-tumor cell subset in the MM BM-TME, which exhibits a high expression of the inhibitory CD161 receptor, binding to the CLEC2D expressed by MM cells, thereby mediating the dysfunction of CD8+ TRMs. Blocking the CD161-CLEC2D pathway is expected to become a novel target for MM immunotherapy. The combination of CD161 inhibitors and CAR-T cell therapy can potentially enhance the anti-myeloma function of CAR-T cells.

Introduction/Background: Bispecific T cell engagers (TCE) targeting BCMA and CD3 induce deep hematologic responses in approximately 60% of heavily pre-treated multiple myeloma (MM) patients. There is an unmet need to overcome primary resistance, as this off-the-shelf therapy is readily available to patients with aggressive disease relapse and has the potential to be delivered in the community. We and others found that high tumor burden leads to primary resistance to TCE 1,2,3,4 and that combination with immunomodulatory drugs (IMiDs) transiently improves responses yet exacerbates T cell exhaustion2. Here, we present preclinical strategies to overcome primary resistance which are tolerable and provide more durable responses specifically for the high tumor burden setting. Methods: We evaluated response and survival to a murine surrogate BCMA/CD3 TCE in the Vk*MYC immunocompetent mouse model of MM expressing human CRBN to investigate tumor burden as a mechanism underpinning primary resistance. We evaluated dosing methods and combination strategies with IMiDs or novel CELMoDs, checkpoint inhibitors, and dexamethasone to improve response rates while maintaining a manageable safety profile. Results: We investigated primary resistance in an expanded panel of Vk*MYC models reflecting differing genomic heterogeneity and confirmed that a threshold of tumor burden limits T cell activation and correlates with resistance to TCE. BCMA is cleaved from the tumor cell surface by gamma-secretase and thus, high density of shed BCMA may act as a sponge to BCMA TCE. However, we found that administration of gamma-secretase inhibitors, while stabilizing BCMA expression on MM cells, is not sufficient to overcome high tumor burden-driven resistance to TCE. We previously reported the combinatorial activity of BCMA TCE with IMiDs and revealed a role for tumor-extrinsic effects of IMiDs in allowing mice with a high tumor burden to transiently respond to BCMA-TCE before T cell exhaustion prevailed2. To build on that combination strategy, we first hypothesized that the addition of a PD1 checkpoint inhibitor could reduce T cell exhaustion that occurs in response the BCMA-TCE in the resistant setting. We found that this triple combination provides more persistent activation and expansion of cytolytic T cells and sustains remission but leads to toxic cytokine release syndrome and early mortality in half of the cohort. Next, to address the acute toxicity from the hyperactivated T cell response and reduce tumor burden, we combined the triple combination with dexamethasone (Dex). To our surprise, concurrent Dex, even in the absence of IMiDs/CELMoDs, boosts responses to TCE. We observe that Dex moderates T cell proliferation in response to the TCE and leads to a moderate sustained cytokine production (IFN-γ, TNF, and IL-2R) systemically and in the bone marrow compared with a high transient burst seen with TCE alone or the concurrent treatment with IMiD + PD1 blockade. We determined that incremental step-up TCE dosing allows the combination of IMiDs or CELMoDs (iberdomide or mezigdomide) with Dex to be completely tolerated. To investigate whether delivering IMiD or CELMoDs before or during TCE administration altered efficacy, we tested pre-treatment with IMiDs/CELMoDs + Dex compared with concurrent dosing and found pre-treatment led to superior durable responses. Pre-treatment with iberdomide + Dex followed by step-up dosed TCE generated 100% response rate and the highest overall survival in subjects bearing a high tumor burden. This was accompanied by a more favorable T cell profiling over time, with limited expansion of regulatory T cells and reduced accumulation of exhausted T cells. Conclusion: Modelling the dosing and combination strategies of a BCMA-TCE in our immunocompetent system has allowed us to understand the dynamic changes in T cell fitness that are required for effective, tolerable, and durable TCE therapy for MM. We illustrate how improvements to TCE therapy mitigate T cell exhaustion and overcome primary resistance, and importantly, suggest a strong immunotherapeutic strategy for MM patients with challenging-to-treat actively progressing disease. 1. Lee et al., Nat Med 29, 2295-2306 (2023) 2. Meermeier et al. Blood Cancer Discov. 2021 Jul;2(4):354-369. 3. Rees et al. Blood Cancer J. 2024 Jul 23;14(1):122. 4. Girgis et al. Blood Adv. 2023 Feb 28;7(4):644-648

Primary resistance to BCMA-directed bispecific antibodies (BCMA-BsAb) is a major clinical hurdle despite their outstanding efficacy for relapsed and refractory multiple myeloma (MM). Primary resistance has been linked clinically to patient characteristics including tumor burden. Our previous work in the immunocompetent Vk*MYC model of MM credentialed that tumor burden also correlates with resistance to a murine BCMA-BsAb, which we addressed through drug combinations with MM backbone agents. Pairing the BsAb with cyclophosphamide (Cy) was well-tolerated, reversed primary resistance, reshaped an immunosuppressive tumor microenvironment (TME) and led to durable responses with immunosurveillance in all but the most aggressive settings. Here, we aimed to improve the BCMA-BsAb and Cy combination strategy with a goal of curing models representing clinically relevant Cy-refractory aggressive settings, by adding T cell checkpoint blockade. Despite a lack of single agent activity of checkpoint inhibitors in MM (and in our models), PD1 and TIGIT are relevant targets as their expression on T cells correlates with both primary and acquired resistance to BCMA-directed cell therapies. Our team hypothesized that blockade of either PD1 or TIGIT with concurrent BCMA-BsAb and Cy would deepen anti-MM responses and provide a more durable immunotherapy regimen by sustaining activation of the T cell repertoire. Methods: We preclinically tested the ability of two weeks of concurrent dosing of BCMA-BsAb with Cy, checkpoint inhibitors targeting either PD1 or TIGIT, or the triple combination to overcome primary resistance and improve survival in a Cy-resistant transplantable Vk*MYC model of MM. We followed the response longitudinally in the blood, and in the bone marrow and spleen after complete responses were achieved at five weeks. Results: The double combination of a checkpoint inhibitor to the BCMA-BsAb was well-tolerated and abolished primary resistance, yet, failed to provide curative responses. Whereas the triple combination with either PD1 or TIGIT blockade remained safe, provided robust initial responses that overcame primary resistance and resulted in durable complete and curative responses in the entire cohort. These survival benefits represent significant improvements over control combinations tested here but also over all historical preclinical anti-MM therapy tested in our models to date. Both PD1 and TIGIT blockade with Cy/BCMA-BsAb triple combinations delivered a similar multiphasic potent anti-tumor response. This response was first characterized by highly effective T cell-mediated lysis of the tumor, as evidenced by deep reduction of M-spike after two weeks of treatment. As we previously published, the addition of Cy to BCMA-BsAb rapidly reshapes the TME by depleting Tregs and exhausted T cells and brings naive T cells to the tumor. However, we observed elevated T cell checkpoint expression at times of BsAb-relapse in aggressive models, suggesting that blockade of these pathways may help sustain T cell activation. Indeed, the ensuing T cell response in the triple combination gradually builds and encourages a pool of less terminally differentiated T cells to proliferate to allow for a more sustained response with repeat dosing. For the first time, we observe that the triple combination significantly engages the CD4 T cell compartment, which can be essential for a complete anti-tumor response. At the time of complete response, single cell analysis displays that T cells retained in the BM were less exhausted and defined by elevated CCR2 expression, a known marker of activated memory T cells, suggestive of ongoing immunosurveillance. Conclusions: Here we demonstrated through controlled in vivo experimentation how the addition of Cy reverts primary resistance to BCMA-BsAb by reshaping the TME in our Cy-insensitive preclinical model. Adding checkpoint inhibitors targeting TIGIT or PD1 further improves the durability of BsAb and Cy combination to cure all mice in these cohorts, by critically lysing the tumor, resetting the suppressive TME, and instigating a gradual, multiphasic, and sustained anti-MM T cell response. The concurrently delivered triple combination is well tolerated and provides optimal temporal control of MM especially in a preclinical model reflecting primary resistance mediated by high tumor burden and may have translational benefit to MM patients.