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Abstract Multiple myeloma (MM) with t(11;14)(CCND1;IGH) remains the only subset sensitive to the BCL2 inhibitor venetoclax. However, not all patients with t(11;14)(CCND1;IGH) respond to treatment, and some progress early after initial response. To investigate this, we examined 44 whole-genome and whole-exome sequencing data samples from 34 patients with t(11;14) MM treated with venetoclax. The presence of mutations in the RAS pathway was strongly associated with shortened progression-free survival (PFS) and was validated in an independent cohort of 21 patients with MM. The presence of 1q gain was also associated with shorter PFS in patients without RAS mutations. In 10 patients with paired prevenetoclax and postvenetoclax treatment samples, postvenetoclax progression was recurrently driven by the selection of genomic events in the BCL2/MCL1 and RAS pathways and of high-risk features (eg, loss of TP53 and CDKN2C). Overall, our study shows that comprehensive genomic profiling can identify most mechanisms underlying resistance to BCL2 inhibition in t(11;14)(CCND1;IGH) MM.

OBJECTIVES: Current research does not address the existence and impact of structured tele-critical care (TCC) training on the delivery of care in the ICU. This pilot study aimed to evaluate the association between on boarding elements focused on training and clinicians perceptions of delivery of care. DESIGN: Cross-sectional survey study. SETTING AND PARTICIPANTS: Critical Care professionals at four U.S. hospitals, professional meetings and Society of Critical Care Medicine’s International Membership base. Participants were active clinicians who practice in the TCC setting. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 432 participants responded to the survey, 227 met the eligibility criteria (52.5%) and were included in the analysis. Respondents were a multi-professional group of TCC clinicians aged between 35–55 years of age (56.4%). Seventy-four percent of respondents reported having orientation before providing TCC, 46% reported having formal mentorship, and 66% reported formal training on their institutional platform. Provision of orientation before participating in a TCC program was associated with higher ratings of feeling prepared (odds ratio [OR], 3.52; p < 0.001) and feeling accepted as part of the ICU team (OR, 2.21; p = 0.008). Mentorship was associated with feeling more prepared (OR, 8.2; p < 0.001) and higher comfort in delivering care (OR, 2.78; p = 0.016). Platform training was associated with feeling more prepared (OR, 4.66; p < 0.001), comfortable in delivering care (OR, 3.6; p = 0.002), feeling accepted as part of the team (OR, 3.18; p < 0.001), and more likely to participate in quality improvement (OR, 2.51; p = 0.001). A site visit also made a positive impact in feeling prepared (OR, 2.86; p < 0.001), comfortable (OR, 4.95; p = 0.002), feeling like recommendations were accepted (OR, 3.73; p < 0.001), more likely to recommend TCC (OR, 3.18; p = 0.001), and participating in quality improvement (OR, 3.24; p < 0.001). CONCLUSIONS: In this pilot study, structured training utilizing orientation, mentorship, and platform training as surrogates, along with a site visit before beginning delivery of care in a TCC setting, were associated with more positive perceptions in the delivery of care domains assessed. We highlight potentially important factors that warrant further evaluation and assessment of the need for standardization across TCC programs.

Abstract Background Ten years after the original publication, the Centiloid framework is now broadly used to harmonize amyloid‐PET quantification, facilitate data sharing and comparison across cohorts, and even assist visual interpretation in clinical settings. We evaluated the global implementation of Centiloids by comparing their distribution and corresponding positivity thresholds across cohorts. Methods We gathered data from publicly available cohorts and reached out to investigators across the world to collect cross‐sectional Centiloids, demographic and clinical information, and visual read data. Gaussian mixture models (GMM, k=2) were fitted to Centiloid values for each cohort and cutoffs were calculated as mean + 2SD of the lower Gaussian. When visual reads were available, we determined Centiloid cutoffs that maximized correspondence with visual reads (Cohen’s kappa). Data was combined across cohorts using random effects meta‐analyses. Results As of January 2025, we included 37 cohorts ( n = 41,678 participants) with heterogeneous pipelines, radiotracers, and clinical and demographic characteristics (Table‐1). The low Gaussian peaks ranged from ‐9 to 10CL; the meta‐analysis identified a common peak at 1CL. The second peaks were more heterogeneous (range=38‐102CL; meta‐analysis outcome=64CL). Across cohorts, the proportion of cognitively unimpaired versus impaired participants impacted the position of both peaks, with better separation in cohorts enriched in impaired individuals (Figure‐1C). The meta‐analysis indicated a GMM‐based cutoff of 19CL (95%CI: 16‐21CL, Figure‐1B); subgroup analyses showed no evidence of significant effect between single versus multicenter settings (17 versus 20CL, p = 0.30), MRI‐based or PET‐only processing (18 versus 19CL, p = 0.88), and no evidence of difference across radiotracers (Flutemetamol: 16CL; PIB: 17CL, Flutafuranol: 18CL, Florbetaben: 19CL, Florbetapir: 20CL, p = 0.78). In a subset of 29,496 participants with visual reads available, binary visual reads corresponded well to Centiloids (common kappa=0.86, Figure‐2A). The visual read‐based cutoff of 24CL (95%CI: 21‐27CL, Figure‐2B) maximized correspondence between visual read and quantification and was slightly higher than the GMM‐based cutoff. All meta‐analysis models showed high non‐random heterogeneity (I 2 >80%) across studies, suggesting non‐random differences in peaks and cutoffs. Conclusions Meta‐analysis‐based cutoffs align well with thresholds from the existing literature. High heterogeneity among studies underscores the need to investigate contributing factors, raising concerns about applying common cutoffs.

Abstract Background Eligibility criteria for clinical trials are crucial for maintaining safety and study integrity. However, overly restrictive criteria can result in unrepresentative trial populations, leading to gaps in understanding real‐world treatment efficacy. Addressing potentially modifiable exclusions (PMEs) could enhance trial accessibility and participation without compromising safety. Methods The authors retrospectively analyzed screen‐failed patients with biliary tract cancer or pancreatic cancer between August 2019 and November 2024 at The University of Texas MD Anderson Cancer Center. Screen‐failed patients were those who provided informed consent but did not participate for any reason. Clinical data were obtained from screening logs and electronic health records. PMEs were identified and validated by two independent medical oncologists. Results Of 585 screen‐failed patients from 18 trials, 509 were analyzed (367 with pancreatic cancer and 142 with biliary tract cancer) after excluding 76 because of incomplete data. Leading causes of screen failure were declined participation (19%), comorbidities (12%), suboptimal organ function (11%), absence of a biomarker (10%), and insufficient biospecimens (7%). Reasons for declining included preference for standard care (23%), travel (22%), and competing trials (5%). The authors identified 69 patients (13.6%) who had PMEs (primarily borderline laboratory abnormalities), including liver function (23%), kidney function (20%), platelet count (12%), hemoglobin (7%), and white blood cell count (6%) abnormalities. Other PMEs included previous or concurrent malignancies (9%) and viral hepatitis (4%). PMEs were evenly distributed across trials. Conclusions Rigid eligibility criteria exclude stable patients who might benefit from investigational treatments. Easing criteria related to incidental or asymptomatic laboratory abnormalities could broaden trial accessibility and improve enrollment in populations that are more representative of real‐world use.

Abstract Background: Routine laboratory testing prior to chemotherapy administration is a cornerstone of hematology and oncology practice, aimed at ensuring the safety and efficacy of treatment. However, over time, standardized protocols may accumulate excessive or low-yield laboratory tests that offer limited value to clinical decision-making. In 2018, Mayo Clinic implemented standardized treatment protocols using Epic Beacon, an electronic health record module that integrates chemotherapy regimens with associated laboratory testing and clinical workflows. While well-intentioned, these protocols may include redundant tests that delay treatment initiation, increase patient discomfort and anxiety, and burden providers with excessive in-basket communications. We hypothesize that many laboratory tests currently embedded within prechemotherapy workflows are unnecessary and can be safely omitted without compromising patient care. Methods: We conducted a retrospective cohort study evaluating the pre-treatment laboratory testing associated with the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy protocol within the Mayo Clinic Rochester Division of Hematology. The study included 100 patients diagnosed with diffuse large B-cell lymphoma who received treatment between January 1 and December 31, 2024. Patients were excluded if they had incomplete medical records or received treatment outside the Mayo Clinic system. Data was abstracted via chart review using the Mayo Data Explorer. We analyzed complete blood counts (CBC), which include 12 individual tests, and comprehensive metabolic panels (CMP), which include 16 individual tests, obtained prior to each treatment cycle. For each cycle, we assessed the proportion of abnormal laboratory results and the proportion of abnormal results deemed clinically relevant which were defined as those that prompted a treatment delay, dose reduction or early termination of a cycle. Results: The cohort comprised 100 patients with a mean age of 68.2 years (SD 13.4) and a median age of 71 years (IQR 58.75–77); 57 were male and 43 were female. Based on available documentation, disease stage was classified as limited (stage I/II; n=41) or extensive (stage III/IV; n=59). A total of 519 R-CHOP cycles were administered, including 419 subsequent cycles (cycles 2–6). Fifteen patients discontinued treatment early, although none of the discontinuations were attributable to abnormalities in pre-infusion laboratory testing. Among the 419 subsequent cycles, 34 (8.1%) experienced treatment delays, with only one delay resulting from a clinically significant laboratory abnormality (hyperglycemia). Additionally, 23 patients underwent regimen modification (dose reduction or drug holding), but none of these were due to pre-infusion lab abnormalities.Of the 411 comprehensive metabolic panels (CMPs) obtained prior to these cycles, 315 (76.6%) contained at least one abnormal value, yielding a total of 786 abnormal results. Similarly, 405 of 415 (97.6%) complete blood counts (CBCs) demonstrated at least one abnormality, generating 2,696 abnormal values in total. Despite the high frequency of abnormal findings, the overall clinical yield was minimal, with only 1 of 826 pre-infusion laboratory panels (0.12%) leading to a modification in clinical management. Conclusion: Routine pre-infusion laboratory testing prior to R-CHOP chemotherapy frequently identifies abnormal results. However, these abnormalities are rarely clinically significant and do not affect treatment decisions. These findings support the reconsideration of current testing protocols to reduce unnecessary testing, enhance workflow efficiency, and minimize patient and provider burden without compromising safety.

Abstract Background: B-cell maturation antigen (BCMA)-targeted T cell engagers (TCEs) have emerged as a highly potent immunotherapy for relapsed/refractory multiple myeloma (MM). However, both primary and acquired resistance significantly limit the durability of responses. Antigen escape and development of dysfunctional T cell responses are a significant driver of relapse post-therapy. Here, we preclinically interrogate if continual versus intermittent/response-adapted dosing differently affects development of resistance, the translatability to clinical resistance mechanisms, and how tumor-intrinsic or microenvironmental factors shape the development of resistance to BCMA-TCEs. Methods: We utilized a syngeneic transplantable Vk*MYC MM model which permits clonal tracking through unique immunoglobulin rearrangements. Two cohorts of tumor-bearing mice were treated with a murine surrogate BCMA-TCE using distinct strategies: (1) continuous weekly dosing until disease progression, and (2) an “ intermittent/response-adapted” strategy, where treatment was paused after initial response and resumed upon detection of M-spike. At time of relapse on therapy, RNA-seq was performed on isolated tumor cells to investigate mutations in- or loss of Tnfrsf17/BCMA. Single-cell RNA sequencing (scRNA-seq) was also performed on 189,025 cells isolated from bone marrow and extramedullary sites. Results: Both continuous and intermittent treatment regimens resulted in similar overall survival. BCMA-TCE treatment induced a distinctive pattern of disease progression, with para- and extra-medullary dissemination observed in 31% (17/54) of treated mice—regardless of treatment schedule. Given this treatment-induced dissemination hasn't been historically observed in our model, we further profiled BCMA expression and infiltrating immune cells to understand relapse mechanisms at these sites. Furthermore, we noted lower BCMA expression in tumors from continuously treated mice compared to untreated or stop-and-go cohorts. Importantly, reduced BCMA expression occurred without loss of plasma cell identity indicating that lineage switching may not be occurring as observed in other hematological malignancies treated similarly (Ruella, 2023, Nat Rev Drug Discov); malignant cells retained high expression of canonical plasma cell markers Irf4, Prdm1, Xbp1, Sdc1. We identified three mechanisms of acquired resistance:Genomic BCMA loss: Complete and irreversible loss of BCMA due to retrotransposition of IAP particles into the first intron of Tnfrsf17, disrupting transcription of downstream exons. This represented a genomic mechanism of antigen escape occurring with continual and prolonged TCE dosing, disrupting transcription and eliminating surface BCMA.Transient BCMA downregulation: Transient downregulation of BCMA expression associated with rapid disease relapse under continuous treatment. Tumors exhibited low BCMA levels and widespread dissemination in paramedullary sites. However, re-transplantation into naïve hosts led to restoration of BCMA expression and re-sensitization to TCE, indicating antigen loss was not permanent.Resistance without antigen loss: In the intermittent dosing regimen, several cases achieved complete remission after two doses, relapsed after treatment pause, and were not controlled after subsequent retreatment. Despite preserved BCMA expression and absence of BCMA mutations or deletions, tumors were refractory to retreatment, suggesting a tumor extrinsic mechanism of resistance. scRNA-seq analysis of the tumor microenvironment is ongoing and the role of uniquely identified immune infiltrates, like NK-like lymphocytes and distinct macrophages, in shaping the cell responses is under consideration. Conclusion: Resistance to BCMA-TCE therapy in the Vk*MYC model of MM arises through multiple mechanisms including genomic antigen loss and transient antigen downregulation. Continuous therapy drives selective pressure leading to target loss via genomic or epigenetic routes, while intermittent dosing preserves antigen but permits resistance to TCE, potentially through immune escape mediated by microenvironmental changes. These insights underscore the translatability of the Vk*MYC model for evaluating rational combination therapies to mitigate these resistance mechanisms. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an IACUC or through an ethical review process.

Abstract Introduction: Despite remarkable advances in treatment, patients with multiple myeloma (MM) continue to suffer relapse. Therefore, the development of novel therapeutic strategies is required. The SWI/SNF chromatin remodeling complex, especially its core ATPase subunits SMARCA2/4, is essential for super-enhancer function and MM cell survival. Furthermore, NSD2, which is overexpressed in t(4;14)-positive MM, has been reported to interact with SMARCA2/4, suggesting differential sensitivity to SMARCA2/4 inhibition. While ATPase inhibitors and PROTAC degraders targeting SMARCA2/4 have demonstrated promising preclinical efficacy in MM and other malignancies, their broader therapeutic relevance remains to be clarified. Methods: To support the clinical development of mSWI/SNF antagonists for MM, we first performed PRISM-based profiling of the anti-proliferative effects of both an mSWI/SNF ATPase PROTAC degrader and a catalytic inhibitor across a panel of 888 cancer cell lines from diverse lineages. Lineage enrichment analysis identified plasma cell myeloma as one of the most sensitive malignancies, with the PROTAC degrader demonstrating particularly potent effects. These were followed by in-depth studies in 35 human myeloma cell lines (HMCLs) using FHD-286 (SMARCA2/4 ATPase inhibitor), AU-24118 (CRBN-based degrader), and AU-15330 (VHL-based degrader). Cell viability was assessed after 5-day treatment by flow cytometry with Annexin V and Live/Dead staining. Additional assays were performed in hCRBN-engineered murine plasmacytoma cell lines and Vk*MYChCRBN MM mouse models. Chromatin profiling employed CUT&Tag with SMARCA2/4 and IKZF1/3 antibodies. Results: FHD-286 and AU-24118 showed potent anti-myeloma activity across most cell lines (median IC₅₀: 4.5 nM and 5.9 nM, respectively). HMCLs with t(4;14), as compared to those without, were more sensitive to FHD-286 (p= 0.0064), with a similar trend for AU-24118 (p=0.09). AU-24118 log2 IC₅₀ values correlated with those of FHD-286 (r=0.62, p=0.0002) and with pomalidomide (r=0.61, p=0.0002), suggesting a shared biology. Indeed, CUT&Tag analysis for SMARCA2/4 and the pomalidomide targets IKZF1/3 showed these factors co-localized at most sites throughout the genome in MM.1S cells, including at DUSP22/IRF4, POU2AF1, and IgH super-enhancers. Treatment with AU-15330, depleted SMARCA2/4 off chromatin at &gt;80% of sites within 8 hours (FDR &lt;0.01), and somewhat surprisingly also depleted IKZF1/3 off chromatin at 70% of bound regions, indicating IKZF1/3 function is dependent upon SWI/SNF. Resultantly, combination studies of SWI/SNF targeting agents and pomalidomide showed a lack of synergy and in some cases antagonism, highlighting the importance of careful evaluation when considering combination with pomalidomide. We and others have demonstrated that murine cells are IMiDs resistant due to species-specific differences in CRBN. Using three distinct mouse plasmacytoma cell lines (+/-hCRBN), hCRBN-expressing cells showed a 100-fold greater sensitivity to AU-24118 compared hCRBN- cell lines. In vivo safety testing of AU-24118 using hCRBN transgenic mice exhibited early mortality within 7 days at doses ≥1.875 mpk and within 3 days at doses ≥7.5 mpk, accompanied by rapid body weight loss. In contrast, a preliminary efficacy study using de novo Vk*MYChCRBN mice, AU-24118 administered at 1 mpk three times per week was well tolerated and led to approximately 40% reduction in serum M-protein within 2 weeks (p&lt;0.05), with no notable weight loss, demonstrating a therapeutic window. Conclusions: Broad PRISM profiling confirms lineage-specific vulnerability of MM to SWI/SNF inhibition, with in vitro and in vivo validation supporting selective efficacy in high-risk t(4;14)-positive subtypes. FHD-286, AU-15330, and AU-24118 demonstrated strong in vitro activity, particularly in t(4;14)-positive HMCLs. This activity was due, in part, to chromatin compaction and decommissioning of lineage and oncogenic enhancers as well as eviction of IKZF1/3 off chromatin. AU-24118 showed high selectivity, with picomolar range IC₅₀ in sensitive HMCLs. Due to species-specific CRBN differences, accurate toxicity evaluation of AU-24118 requires hCRBN-expressing models, and preliminary data indicate in vivo efficacy in the highly predictive Vk*MYChCRBN model. These findings support the potential of SMARCA2/4 targeting agents, especially AU-24118, as promising therapies for MM, particularly in high-risk t(4;14)-positive cases.

Abstract Background Central nervous system involvement in multiple myeloma (CNS-MM) is a rare but highly aggressive manifestation. Limited data exists regarding its incidence, biology, treatment patterns, and outcomes. A deeper understanding of the features unique to CNS-MM will help inform risk stratification and management strategies. Methods We conducted a retrospective study of CNS-MM patients at our institution between January 2000 and December 2023. We identified 305 MM patients with pathology-proven extramedullary disease (EMD). CNS involvement was defined as the presence of parenchymal (brain or spine) or leptomeningeal involvement by MM. Univariate analysis was performed to compare the clinical characteristics between EMD patients with and without CNS-MM. Results Out of 305 patients with EMD, we identified 22 patients with CNS-MM. Common symptoms prompting evaluation of CNS-MM were headaches (32%), numbness or tingling (32%), weakness (27%), confusion (23%), falls (23%), and visual disturbances (14%). Among these patients, 17 (77%) had detectable disease in cerebrospinal fluid (CSF), 12 (55%) in the brain parenchyma, 4 (18%) in the spinal cord parenchyma and 6 (27%) in the leptomeninges. Of the CNS-MM patients, the median age at diagnosis of MM was 58.3 years and the median time to CNS-MM involvement was 16.2 months, with 3 (14%) patients having CNS involvement at diagnosis. The predictors of CNS-MM development were high-risk cytogenetics (OR 2.9, p=0.04) and presence of additional visceral organ involvement outside of the CNS (OR 3.4, p=0.03). EMD was multifocal in 20 CNS-MM patients (83%) and visceral organ involvement [most commonly lymphadenopathy (27%), solid organ (18%) and pleural fluid (14%)] was present in 14 (64%) of these patients, compared to 54% in EMD patients without CNS-MM. High-risk cytogenetics [del(17p), 1q gain/amplification, t(4;14), t(14;16), t(14;20)] were seen in 16 (73%) patients, most commonly 1q gain or amplification (40%), t(4;14) (23%), and del(17p) (18%). Five (23%) exhibited a double-hit profile. In comparison, 113 (40%) EMD patients without CNS-MM had high risk cytogenetics. The median overall survival (OS) from diagnosis of CNS-MM was 6 months (95%CI:1.26-36) compared to 15.6 months (95%CI: 12-18) from diagnosis of EMD in EMD patients without CNS-MM (p=0.05). Median OS from diagnosis of MM in the CNS-MM cohort was 36 months (95%CI: 22.8-NA) compared to 55.2 months (95%CI: 45.6-68.4) in EMD patients without CNS-MM (P=0.11). When excluding CAR-T or bispecific therapy recipients, median OS from diagnosis of MM for CNS-MM was 22 months versus 48 months in EMD patients without CNS-MM (P=0.02). Overall response rate in our CNS-MM cohort with first treatment for EMD was 32% compared to 48% in EMD patients without CNS-MM (p=0.04). In patients with CNS-MM, median progression-free survival (PFS) from CNS-MM diagnosis was 3.3 months (95%CI: 0.8-12) versus 3.9 months (95%CI: 3-5.1) in EMD patients without CNS involvement (p=0.2). Six (27%) CNS-MM patients received CAR-T; 3 received cilta-cel, 2 received ide-cel and one received a BCMA CAR-T on trial. Bispecific therapy was used in 4 (18%) patients, with 2 receiving Teclistamab and 2 Talquetamab. Three (14%) CNS-MM patients received both CAR-T and bispecific therapy. The median PFS on CAR-T therapy was 10.5 (95%CI: 5.6-NA) months and 12.7 (95%CI: 10.2-NA) months on bispecific therapy. The median OS from diagnosis of CNS-MM for patients who received CAR-T or bispecific therapy was 36 months (95%CI: 14.0-NA), compared to 1.2 months (95%CI: 0-14.4) for those who did not (p=0.002). Six (27%) patients with CNS-MM, all of whom had CSF involvement of disease, received intrathecal therapy; methotrexate in 3, cytarabine in 2, and one receiving both. Two of these patients demonstrated improvement in cerebrospinal fluid findings following intrathecal chemotherapy, in conjunction with other therapies. Patients who received intrathecal chemotherapy had a median OS measured from development of EMD of 10.8 (95%CI: 3.6-NA) months, compared to 15 months (95%CI: 4.8-NA) in those without IT chemotherapy (p=0.59). Conclusion Central nervous system involvement in MM is relatively uncommon and associated with dismal outcomes. High-risk cytogenetics and involvement of other visceral sites are associated with a higher risk of CNS involvement. Intrathecal therapy offers limited value and immune effector therapies represent efficacious options for treatment.

Abstract Introduction High disease burden has been associated with reduced efficacy and resistance to BCMA CAR-T therapy in relapsed/refractory multiple myeloma (van de Donk, Blood Cancer Discov, 2021; Martin, J Clin Oncol, 2023). While CAR-T cells can still induce remissions in these patients, outcomes are often worse compared to those with a lower tumor load. In addition to tumor debulking, novel strategies are needed to improve responses and durability of CAR-T cells in these patients. We previously demonstrated that immunomodulatory drugs (IMiDs) improve in vivo responses to T cell engagers (TCE) in high tumor burden setting by boosting T cell activation (Meermeier, Blood Cancer Discov, 2021; Meermeier, Blood, in press). IMiDs have also been shown to promote CAR-T cell antitumor activity in moderate to low tumor burden conditions (E:T ratios ≥1:1) (Yan, J Transl Med, 2024; Wang, Clin Cancer Res, 2018). We hypothesized that combining CAR-T with an IMiD may improve CAR-T function in high tumor burden setting. Methods We generated murine anti-BCMA.CD28 CAR-T cells by transducing Con-A stimulated T lymphocytes extracted from hCRBN+ mice, expanded with IL-7 and IL-15 to express a memory phenotype. We then tested CAR-T, or untransduced T cells, ability to kill in vitro IMiD resistant, hCRBN-, Vk*MYC myeloma cells in low (E:T 1:1) and high (E:T 1:≥5) tumor burden, with and without pomalidomide (POM) and IL-2. Tumor killing and T cell phenotype were assessed by flow cytometry at 24-72 hours. This controlled system allowed us to focus on putative CAR-T cell intrinsic impact of IMiDs. Results In low tumor burden setting, CAR-T effectively killed tumor at all timepoints, with or without POM; addition of POM increased T cell number, confirming that IMiDs support CAR-T expansion. Unexpectedly, in high tumor burden (E:T 1:≥5), adding POM diminished CAR-T mediated killing over time and hindered CAR-T expansion. Moreover, combination with POM decreased granzyme B, while increased expression of exhaustion marker TOX, ultimately leading to CAR-T apoptosis. This phenotype was further intensified by increasing concentrations of IL-2 in the co-culture, resulting in increased expression of CAR-T expressing LAG-3, TIM-3, and TIGIT, suggesting that CAR-T cells may be overactivated by POM and IL-2. Our manufacturing protocol preferentially expands memory CAR-T cells, favored for their durability, proliferative capacity, and considered to rapidly expand upon encounter with antigen. We reasoned that CAR-T are already sufficiently activated by CAR/CD3z stimulation, especially with a CD28 co-stimulatory domain (versus 4-1BB), known promote rapid, effector memory expansion upon activation (Kawalekar, Immunity, 2016). Activation is intensified by high tumor burden where antigen is highly abundant. We hypothesize under intense, sustained antigen exposure in high tumor burden, the addition of POM paradoxically triggers activation induced cell death (AICD). Conclusion Although it is well accepted that POM can boost T cell function and proliferation, we demonstrate that in high tumor burden, POM drives memory CAR-T to increase exhaustion markers and apoptotic cell death by overactivation, further exacerbated by exogenous IL-2. Interestingly, we have shown opposite effects of preclinically combining POM with TCE therapy in high tumor burden, based on dosing schedule. Concomitant administration transiently boosted T cell activation improving response rates but rapidly led to T cell exhaustion (Meermeier, Blood Cancer Discov, 2021). In contrast, IMiD pretreatment in the context of step-up dosing TCE administration improved T cell fitness and longevity (Meermeier, Blood, in press).The effect of IMiDs on T cells transcriptional program is likely to differ depending on differentiation stage and mode of T cell activation. Memory cells have more sensitive TCR signaling (Kumar, Immunity, 2011) and can be hyperactivated, leading to AICD. Thus, timing of IMiD administration, when tumor burden is low, may be a critical consideration as different T cell subsets require different levels of activation. Conversely, TCE provide only initial activation via TCR signaling, therefore POM may help in providing necessary co-stimulation in this setting. Future studies will evaluate different co-stimulatory domains and test in vivo effects of POM on CAR-T expansion and persistence, particularly as CAR-T cells diminish over time as tumor burden increases.

Abstract Introduction Venetoclax combined with hypomethylating agents (VEN/HMA) is an approved frontline induction therapy for older or unfit patients with acute myeloid leukemia (AML). However, data comparing its efficacy and safety to intensive chemotherapy (IC) in intermediate-risk AML—characterized by molecular and cytogenetic heterogeneity—remain limited. Methods This multicenter, real-world retrospective study analyzed newly diagnosed intermediate-risk AML patients who received either VEN/HMA or IC. We evaluated composite complete remission (CRc) rates, transfusion and infection rates, long-term outcomes including overall survival (OS) and progression-free survival (PFS), and performed subgroup analyses to identify mutation-specific predictors of treatment response. Multivariable Cox regression was used to identify independent prognostic factors. Results A total of 229 intermediate-risk AML patients were included. VEN/HMA achieved a comparable CRc rate to IC (75.3% vs. 63.4%) with lower transfusion needs and fewer infections. Patients harboring FLT3-ITD, DNMT3A, or TET2 mutations showed greater benefit from VEN/HMA, while KIT-mutated patients responded more favorably to IC. Among patients who did not undergo allo-HSCT, VEN/HMA significantly improved 3-year OS (P = 0.001) and PFS (P = 0.008). No significant OS or PFS difference was observed in the allo-HSCT subgroup. In MRD-negative patients without transplant, those treated with VEN/HMA had superior OS compared to IC. Multivariable analysis identified VEN/HMA (HR = 0.613, P = 0.038) and allo-HSCT (HR = 0.11, P = 0.001) as independent predictors of better survival. Conclusions VEN/HMA is a safe and effective induction strategy for intermediate-risk AML, particularly in patients with BCL-2 inhibitor–sensitive mutations (FLT3-ITD, DNMT3A, TET2). These findings support a genotype-guided treatment approach in AML, complementing current risk stratification frameworks and informing individualized induction therapy decisions.