Efficacy and durability remain central shortcomings of T-cell based therapies in multiple myeloma (MM). Here, we employ blood-based transcriptional T-cell profiling to define impaired T-cell fitness as putative biomarker associated with sensitivity to PD1 inhibition in CAR-T refractory MM patients.

Although most patients with multiple myeloma respond to treatment initially, therapy resistance develops almost invariably, and only a subset of patients show durable responses to immunomodulatory therapies. Although the immune microenvironment has been extensively studied in patients with myeloma, its composition is currently not used as prognostic markers in clinical routine. We hypothesized that the outcome of immune signaling pathway engagement can be highly variable, depending on which 2 cellular populations participate in this interaction. This would have important prognostic and therapeutic implications, suggesting that it is crucial for immune pathways to be targeted in a speci fic cellular context. To test this hypothesis, we investigated a cohort of 25 patients with newly diagnosed multiple myeloma. We examined the complex regulatory networks within the immune compartment and their impact on disease progression. Analysis of immune cell composition and expression pro files revealed signi ficant differences in the Bcell compartment associated with treatment response. Transcriptional states in patients with short time to progression demonstrated an enrichment of pathways promoting B -cell differentiation and in flammatory responses, which may indicate immune dysfunction. Importantly, the analysis of molecular interactions within the immune microenvironment highlights the dual role of signaling pathways, which can either be associated with good or poor prognosis depending on the cell types involved. Our findings therefore argue that therapeutic strategies targeting ligand-receptor interactions should take into consideration the composition of the microenvironment and the speci fic cell types involved in molecular interactions.

Introduction In the times of promising immunotherapies against multiple myeloma (MM), close monitoring of patients' response and disease complexity is crucial for accurate decision making. To date, bone marrow (BM) aspiration and biopsy have been the gold standard for diagnosis and molecular characterization of the disease. These procedures, however, are invasive and associated with discomfort for the patients. Not all existing disease clones can be captured by a one-time aspiration at a single location. Furthermore, current standard diagnostic methods such as FiSH and bulk DNA sequencing provide little information with regard to decision making about treatment with modern therapeutics such as monoclonal antibodies, bispecific T-cell engagers, and CAR-T cells. Here, we present data to show that single-cell RNA sequencing (scRNA-seq) of circulating MM cells is an excellent proxy for detecting prognostically relevant inferred copy number variants (CNVs) and translocations, while depicting the heterogeneity of the disease. Moreover, scRNA-seq can yield additional, therapeutically relevant information such as expression level of the target protein for the immunotherapies. Because of the non-invasiveness and possibility of frequent peripheral blood (PB) draws, this method constitutes a great tool for patient monitoring in both clinical-trial and non-trial settings. Methods Fresh PB and BM samples of 10 myeloma patients at various disease stages were obtained. Mononuclear cells were isolated by density-gradient centrifugation. CD138-positive cells were selected using magnetic-beads separation. Single MM cells were sorted onto 96-well plates using the following gating strategy: 7AAD-, CD14-, CD138+, CD38+, SLAMF7+, CD45-/CD45dim. RNA isolation and cDNA-library preparation were performed using the Smart-seq2 method (Picelli et al. Nat Prot 2014), which yielded information of the full transcript length. For testing purposes, we used our previously published data (Frede et al. Nat Cell Biol 2021). Clustering was performed with Seurat. Cell-type annotation was performed using SingleR and reference dataset from the BLUEPRINT consortium. Light- and heavy-chain B-cell receptor sequences were determined using BASIC and igBLAST. CNV analysis was performed with inferCNV. Expression level was assessed for genes expected to be overexpressed in case of translocations (CCND1, CCND3, MAF, MAFB, MMSET, FGFR3) as well as for surface marker genes (CD138, CD38, SLAMF7, BCMA, GPRC5D, FCRH5). Fisher test (expression of a particular gene vs. all other genes without the gene of interest) was performed to define the p-value cut-off for identifying an inferred translocation. Percentage of MM cells expressing a surface marker was assessed for each sample. Results We could reproduce the heavy- and light-chain clonality with a sensitivity and specificity of 100 %. scRNA-seq showed to be a valid substitute for BM FiSH with a sensitivity of 94.4 % and specificity of 100 % in detecting prognostically relevant CNVs (amp/gain(1q), del(1p), del(17p), hyperdiploidy) and translocations (t(11;14), t(6;14), t(14;16), t(14;20), t(4;14)). Interestingly, we could show a direct correlation between very low expression of BCMA (expression in 8.1% of circulating MM cells) 29 months after 1st CAR-T cell therapy and progressive disease directly after re-exposure to a 2nd dose of anti-BCMA CAR-T cells (31 months after the 1st administration), suggesting that circulating tumor cells may aid in identifying effective therapies and avoiding those that are not. Furthermore, we observed 2 patients with significant downregulation of BCMA 1.5 months and 14 months (10.5 % and 3 %, respectively) after disease progression under belantamab mafodotin as well as a primary low expression (8.6 %) of FcRH5 in a patient naïve to anti-FcRH5 treatment. Moreover, in 2 patients, FiSH from BM was not feasible due to technical difficulties, which further highlights the utility of interrogation of single MM cells from PB. Conclusion scRNA-seq of MM cells from PB is a robust, non-invasive substitute for BM FiSH to detect prognostically and therapeutically relevant CNVs and translocations. Furthermore, it yields additional information such as expression level or absence of surface markers as immunotherapeutic targets. This is of critical use in clinical decision making and provides opportunity for more personalized therapy approaches.

Metabolic flux analysis in U266 and DP6 single-cell clones

Transcriptional activation of oxidative phosphorylation pathway genes in DP6 dabrafenib-persistent cells

Rotenone sensitivity in dabrafenib-persistent vs. naïve single-cell clones

Antibodies used for Western Blot experiments

Background: Cell-based immunotherapies, particularly chimeric antigen receptor modified (CAR-) T cells, have produced impressive responses in a subset of patients with hematological malignancies. However, the limited durability of responses and the development of treatment resistance present significant challenges. Limited expansion and persistence of CAR-T cells, T-cell exhaustion, and a hostile immune microenvironment are key factors that restrict the efficacy of CAR-T cell therapies. Aims: To enhance the efficacy of CAR-T cell therapy and overcome treatment resistance, a better understanding of their co-evolution with the immune microenvironment and their reciprocal interactions is needed. Therefore, this study aimed to define alterations in cellular states in CAR-T cells and the immune compartment upon BCMA CAR-T cell therapy in relapsed/refractory multiple myeloma (RRMM) patients at single cell resolution. We further aimed to investigate cell-cell interactions and identify novel immunomodulatory pathways for the optimization of T cell-based cancer therapies. Methods: We performed droplet-based single-cell RNA sequencing of CD45+ immune cells from the bone marrow of 4 RRMM patients receiving BCMA-directed CAR-T cell therapy (KarMMa-2 trial; NCT03601078) in combination with immune profiling and VdJ sequencing, before as well as one month and six months after CAR-T infusion. We further performed longitudinal full-length single cell RNA-sequencing of CAR-T cells and CD8+ T cells isolated from the peripheral blood and bone marrow of a total of 24 patients (on KarMMA-2/KarMMa-3 trials; NCT03651128). Results: We defined compositional changes in the bone marrow immune microenvironment upon CAR-T cell therapy and observed a temporary expansion of the myeloid compartment at one month following CAR-T infusion. However, we detected the greatest relative changes in abundance in the CD8 T cell compartment, with an expansion of GZMB+ CD8 effector T cells and a decrease in CD8 naïve and CD8 memory T cell populations over time. We were able to show that CAR-T cell treatment drives bystander CD8+ T lymphocyte differentiation, which initially supports a clonal anti-tumor immune response, but ultimately results in T cell exhaustion, reduced potential for self-renewal and sustained depletion of the tumor-reactive T cell repertoire. We observed an expansion of pre-existing T cell clones six months after CAR-T infusion (P = 0.00018 by paired Wilcoxon test) and found that these undergo terminal differentiation. We predict diminished responses to further immunotherapies, such as response to checkpoint blockade. To investigate how exhaustion is regulated, we analyzed the transcriptional modules active in different cell states and identified TCF7, IKZF1 and PRDM1 among the transcription factors regulating divergent cell states. Inferring cell-cell interactions, we identified crucial interactions of the cell-cell communication network that contribute to T cell exhaustion and regulate immune capacity. Summary/Conclusion: The ability of the CAR-T cells to shape a regulatory immune microenvironment may explain why repeated immunotherapies become increasingly less successful, even when targeting distinct antigens. Our studies therefore have important implications for the timing and sequencing of immunotherapies. Our studies provide a framework for assessing and manipulating the ‘mileage’ of the immune system as a predictive marker and a therapeutic opportunity. Keywords: Myeloma, Cancer immunotherapy, T cell response, CAR-T

Copy number variations in dabrafenib-resistant U266 single-cell clones as compared to baseline

Transcriptional adaptation in DP6 persistent cells

Dabrafenib sensitivity in DP6 single clones

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

Annotation of single cells based on origin

IACS-010759 sensitivity in dabrafenib-persistent vs. naïve single-cell clones

Dabrafenib sensitivity in dabrafenib-resistant U266 single-cell clones as compared to baseline

Background: Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy in children and young adults that frequently becomes treatment-refractory and relapses. Oncogenic NOTCH signaling is a major driver of T-ALL resulting in malignant transformation of T cell progenitors in the bone marrow. With over 50% of the patients harboring gain-of-function NOTCH mutations, inhibiting the oncogenic signaling presents an attractive targeting strategy, yet patient response is often short-lived and current treatment options for relapsed/refractory disease are of limited success. Existence of epigenetically and transcriptionally distinct cell subpopulations has been recently suggested as driver mechanism for treatment escape in refractory T-ALL. Aims: To characterize cell states and the transcriptional changes mediating state transitions as an explorative strategy to develop targeted therapies overcoming drug resistance in T-ALL. Methods: We performed full-length single-cell RNA sequencing of 3,188 leukemia cells collected from the blood of 2 sensitive and 3 refractory PDX models carrying activating NOTCH1 mutations, before and after treatment with the NOTCH inhibitor DBZ (Dipenzazepine). To characterize transcriptional states, we assessed developmental potential using CytoTRACE and identified state-specific gene-regulatory networks (GRN) driving state transition using SCENIC. Alterations in 3D chromatin structures differentiating sensitive and refractory models were assessed by evaluating enhancer rewiring and mapping of the associated loops using Hi-ChIP with an antibody against H3K27ac. Apoptotic priming and functional validation of anti-apoptotic dependencies were determined by BH3 profiling. Results: Analysis of single cell profiles of refractory T-ALL revealed dramatic transcriptional reprogramming resulting in expression of immature hematopoietic signatures and lineage infidelity co-existing within individual leukemia cells. Assessment of developmental programs prior to treatment indicated that deranged lineage commitment can predict response to NOTCH inhibition in vivo. Upon treatment, we observed expansion of 2 distinct populations that critically differ in differentiation stage and developmental trajectory. Fast-cycling cells express progenitor signature and genes associated with RNA processing and slow-cycling cells are enriched in lymphoid differentiation programs. Further analysis of developmental potential revealed that loss of active regulons and gain of transcriptional dependencies underlie the differentiation state transitions and treatment refraction. We identified ATF4 as a key transcriptional dependency in immature cell states. Assessment of epigenetic rewiring underlying cell state transitions demonstrated that immature states present increased numbers of enhancer-promoter interactions driving transcriptional plasticity. Enhancer rewiring results in the emergence of transcriptional dependencies leading to perturbed apoptotic signaling, including a dependency switch from BCL2/BCLxL to MCL. Summary/Conclusion: In conclusion, single-cell transcriptomic analysis identified cell states with high plasticity that are characterized by aberrant differentiation trajectories, distinct transcriptional circuitries and enhancer rewiring resulting in treatment escape. Definition of state-specific transcriptional dependencies combined with BH3 profiling could predict response to NOTCH inhibition and allowed the identification of potential therapeutic targets to overcome Notch-inhibitor resistance in T-ALL. Keywords: Drug resistance, Plasticity, T-ALL, RNA-seq

Cellular plasticity has recently emerged as an underlying mechanism for treatment refraction in cancer. Hence, characterizing cell states and the transcriptional changes required for state transitions is critical for the development of efficient targeted therapies. Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. Using T-ALL patient-derived xenograft (PDX) models carrying activating NOTCH1 mutations, we aim to u First, to determine how cell state diversity drives treatment refraction in T-ALL, we performed full-length single-cell transcriptome analysis on 3188 immune cells collected from 2 sensitive and 3 refractory PDX models. PDX models were treated with either with NOTCH inhibitor DBZ (Dipenzazepine; 10 μM/kg every other day intravenously) or vehicle. Profiled cells were collected at both a short (5 days) and a late time point (x-150 days) post-treatment to assess the effects of DBZ. Analysis of early hematopoietic and thymic precursor signatures demonstrate expression of immature hematopoietic signatures and acquisition of alternative lineage identities coexisting within the same cell, predominantly in refractory models. Analyses of pre-treatment samples indicated that disruption of the normal developmental hierarchy was associated with blockade at an early stage of T-cell commitment and can predict response to NOTCH inhibition in vivo. Upon treatment, cells underwent dramatic transcriptional reprogramming resulting in expansion of 2 distinct populations that greatly differ in both differentiation stage and developmental trajectory. One population expresses lymphoid differentiation programs, whereas the other population is enriched in genes associated with RNA processing and more immature hematopoietic signatures. Cell fate trajectories assessment using Monocle 2, revealed that consistent with their immature hematopoietic signature, refractory cells map at the start point of the differentiation trajectory, while sensitive cells clustered at the endpoint. We next sought to characterize the differentiation hierarchies that define the immature states. Developmental potential as assessed by CytoTRACE was higher in refractory cells when compared to their sensitive counterpart. To identify determinants of cell fate in a functionally relevant manner, we inferred gene regulatory network configurations using SCENIC and defined state specific regulon activity. Among the most prominently expressed regulons present in mature states, we identified transcriptional regulators involved in T-cell differentiation such as IKAROS, GATA3, and SOX5, while in immature states we observed high transcriptional regulon activity in transcription factors associated with renewal of hematopoietic progenitors such as ATF4, BCLAF1, and MYC in addition to the chromatin remodelers SMARCA4 and EZH2. To assess whether transcriptional rewiring in refractory cells results from greater chromatin accessibility, we performed single-cell ATAC sequencing (10x Genomics) on splenic cells from 1 sensitive and 1 refractory T-ALL PDX model treated with DBZ or vehicle. After integration with corresponding single-cell RNA-sequencing data, annotated ATAC profiles demonstrated greater chromatin accessibility, predominantly in active promotor and enhancer regions, in the refractory model compared to the sensitive model. Peak to gene linkage analysis revealed widespread alterations in cis-regulatory regions near genes associated with immature hematopoietic precursor signatures such as CD33. Furthermore, greater chromatin accessibility in refractory cells was associated with an increased number of enhancer loops per expressed gene as determined by H3K27ac HiChIP analysis. In conclusion, we demonstrated that presence of highly plastic cellular states defined by aberrant differentiation trajectories, distinct transcriptional circuitries, and remodeled chromatin architecture results in treatment escape in T-ALL.

Interrogation of cell-free DNA (cfDNA) represents an emerging approach to non-invasively estimate disease burden in multiple myeloma (MM). Here, we examined low-pass whole genome sequencing (LPWGS) of cfDNA for its predictive value in relapsed/ refractory MM (RRMM). We observed that cfDNA positivity, defined as ≥10% tumor fraction by LPWGS, was associated with significantly shorter progression-free survival (PFS) in an exploratory test cohort of 16 patients who were actively treated on diverse regimens. We prospectively determined the predictive value of cfDNA in 86 samples from 45 RRMM patients treated with elotuzumab, pomalidomide, bortezomib, and dexamethasone in a phase II clinical trial (NCT02718833). PFS in patients with tumor-positive and -negative cfDNA after two cycles of treatment was 1.6 and 17.6 months, respectively (HR 7.6, P < 0.0001). Multivariate hazard modelling confirmed cfDNA as independent risk factor (HR 96.6, P = 6.92e-05). While correlating with serum-free light chains and bone marrow, cfDNA additionally discriminated patients with poor PFS among those with the same response by IMWG criteria. In summary, detectability of MM-derived cfDNA, as a measure of substantial tumor burden with therapy, independently predicts poor PFS and may provide refinement for standard-of-care response parameters to identify patients with poor response to treatment earlier than is currently feasible.

Background: Idecabtagene Vicleucel (ide-cel) is the first FDA-approved BCMA-directed CAR T cell therapy for the treatment of relapsed/refractory multiple myeloma (RRMM). Despite remarkable activity of 85% objective response rate and a progression-free survival (PFS) of 11.8 months reported in the CRB-401 trial (NCT02658929), most responses to ide-cel are not durable, and nearly all patients (pts) eventually relapse, even those achieving MRD-negative complete remission (CR). Factors associated with clinical relapse to ide-cel include BCMA loss, lack of CAR T persistence, T-cell exhaustion and PD-1/ PD-L1 impaired T-cell fitness. Here, we report clinical data and correlative findings for 4 pts who were salvaged with a nivolumab (anti-PD1)- based triplet therapy at the time of clinical relapse to first or second ide-cel infusion. Methods: Four pts with penta-refractory RRMM were treated as part of the CRB-401 phase 1 study as previously reported (Raje et al.N Engl J Med.2019;380:1726-37). All pts received nivolumab along with lenalidomide-dexamethasone (Nivo-Rd) or pomalidomide-dexamethasone (Nivo-Pd) at the time of relapse to ide-cel. CD3+ T cells (n=1,047) were isolated from the peripheral blood of 2 of these pts and full-length single-cell RNA sequencing (scRNA-seq) was conducted before and upon treatment with Nivo-Rd/-Pd. Response rates were assessed as per IMWG criteria. Results: The median age of pts was 48.5 years (range 37-56). Two out of 4 pts had cytogenetic high-risk MM and all pts were penta-refractory at the time of CRB-401 study inclusion. The median PFS and OS after first ide-cel infusion were 10.4 (range 3.0-13.8) months and 21.5 (range 16.0-34.6) months, respectively. Three pts received a second ide-cel infusion, whereof only 1 pt achieved a partial response (PR) at a PFS of 5.2 months. Two pts each were treated with Nivo-Rd and Nivo-Pd respectively at the time of progression to ide-cel. Responses to anti-PD1- based therapy varied markedly with 1 PR, 1 stable disease (SD) and 2 progressive disease (PD) as per IMWG criteria. The median PFS to nivolumab-based salvage therapy was 3.6 (range 0.4-8.1) months. Interestingly, pts MM1 and MM2 both had a CR to first ide-cel infusion (PFS 13.8 and 11.9 months, respectively), but while both pts did not respond to second CAR T cell infusion, the benefit from anti-PD1 treatment differed markedly in both pts. MM1 reached a PR and PFS of 105 days. In contrast, MM2 experienced immediate PD and succumbed to his disease 3.0 months later. Profiling of CD3+ T cells from MM1 and MM2 by scRNA-seq revealed rapid cellular state changes in response to PD1 inhibition. PAGODA2 clustering and t-Stochastic Neighbor Embedding (t-SNE) visualization revealed 3 distinct clusters, segregating to an individual pre-anti-PD1 cluster for MM1 and MM2 each and a shared post-anti-PD1 cluster comprising CD3+ T cells from both pts. The pre-anti-PD1 cluster for MM1 contained CD3+ T-cells with a memory T cell- like phenotype (IL7Rhigh, CD62Lhigh) and decreased expression of markers correlating with T-cell fitness (GNLY, PRF1, NKG7, GZMH,LY6C, KLRG1). This phenotype could be rescued, and T-cell fitness could be restored by Nivo-Pd treatment, potentially contributing to the clinical response observed in MM1. Opposingly, the pre-anti-PD1 cluster for MM2 showed intact T-cell fitness with low expression of T-cell exhaustion markers. No clinical benefit to Nivo-Rd was noted in this pt. Conclusions: Checkpoint inhibition and IMiD therapy can induce clinical responses at the time of relapse to first or second CAR T infusion. Efficacy however seems to depend on impaired T-cell fitness as premise for PD1 inhibition to re-induce clinical responses in selected pts.

Supplementary Data from Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy