Supplementary Data containing the statistical analysis plan, Supplemental Figures 1-6 and Supplemental Tables 1 and 2

Introduction Proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) reflect synergistic treatment modalities in multiple myeloma (MM) and their combination marks the backbone of approved first line therapies. Although clinically established, the molecular mechanisms, how IMiDs convey synergistic anti-myeloma activity in combination with PIs, remain elusive. The currently most anticipated mode of action of IMiDs is the redirection of the CRBN E3 ubiquitin ligase towards the transcription factors IKZF1/3 leading to their ubiquitin-dependent degradation, which creates a paradoxon as the combination with PIs would be expected to antagonize this effect of IMiDs by preventing proteasomal degradation of those CRBN neosubstrates. Previously, we described CRBN as an HSP90 co-chaperone with a central role in the quality control of transmembrane proteins, a function compromised by IMiDs mediating anti-MM activity. Against this background, we investigated whether excessive accumulation of unfolded proteins and the induction of the unfolded protein response (UPR) contributes to the synergism of PIs and IMiDs. Methods Different MM cell lines as well as primary patient-derived CD138+ MM cells extracted from newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM) patients were exposed to single agent treatment with bortezomib (PI), lenalidomide (IMiD) or a combination of both. Functionally, UPR signaling was studied on transcriptional and on protein level using qPCR and Western Blot analyses. The intracellular accumulation of misfolded proteins was analyzed by FACS and confocal microscopy. Results We subjected IMiD-sensitive and -resistant MM cell lines as well as primary patient-derived CD138+ MM cells to single treatments with bortezomib, lenalidomide or the combination of both. Bortezomib treatment, as expected, triggered an acute activation of the UPR. Strikingly, we also observed UPR induction upon single lenalidomide exposure. This effect was significantly enhanced upon the combination of both agents as evidenced by intracellular accumulation of misfolded proteins, phosphorylation of ER stress sensors and activation of UPR-specific transcriptional signatures, ultimately resulting in increased induction of intrinsic apoptosis. This suggests that the synergistic anti-myeloma activity of PIs and IMiDs arises from the collaborative activation of the UPR through the simultaneous disruption of the HSP90 co-chaperone function of CRBN and the protein degradation machinery. Of note, the observed UPR phenotype upon lenalidomide treatment was neither evident in IMiD-resistant MM cell lines nor RRMM patients. To systematically validate these findings, we set up a multi-omics screening approach (RNASeq and mass spectrometric analysis) comparing differentially expressed genes or proteins upon lenalidomide treatment in an IMiD-sensitive or -resistant MM setting. Conclusion We propose that the synergism of PI and IMiDs in MM patients relies, at least in part, on the collaborative effect of both drugs on the induction of toxic protein aggregates and the activation of the UPR.

B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.

Biomarkers that predict response to lenalidomide maintenance therapy in patients with multiple myeloma (MM) have remained elusive. We have shown that immunomodulatory drugs (IMiDs) exert anti-MM activity via destabilization of MCT1 and CD147. In this study, cell samples of 654 patients with MM who received lenalidomide (n = 455), thalidomide (n = 98), or bortezomib (n = 101) maintenance were assessed by gene expression profiling and RNA sequencing, followed by correlation of MCT1 and CD147 expression with data for progression-free survival (PFS) and overall survival (OS). Patients with high expression levels of MCT1 showed significantly reduced PFS (31.9 months vs 48.2 months in MCT1high vs MCT1low; P = .03) and OS (75.9 months vs not reached [NR] in MCT1high vs MCT1low; P = .001) in cases with lenalidomide maintenance, whereas MCT1 expression had no significant impact on PFS or OS in cases with bortezomib maintenance. We validated the predictive role of MCT1 for IMiD-based maintenance in an independent cohort of patients who received thalidomide (OS, 83.6 months vs NR in MCT1high vs MCT1low; P = .03). Functional validation showed that MCT1 overexpression in human MM cell lines significantly reduced the efficacy of lenalidomide, whereas no change was observed with bortezomib treatment, either in vitro or in a MM xenograft model. Our findings have established MCT1 expression as a predictive marker for response to lenalidomide-based maintenance in patients with MM.

Abstract Personalized medicine aims to match the right drug with the right patient by using specific features of the individual patient's tumor. However, current strategies of personalized therapy matching provide treatment opportunities for less than 10% of patients with cancer. A promising method may be drug profiling of patient biopsy specimens with single-cell resolution to directly quantify drug effects. We prospectively tested an image-based single-cell functional precision medicine (scFPM) approach to guide treatments in 143 patients with advanced aggressive hematologic cancers. Fifty-six patients (39%) were treated according to scFPM results. At a median follow-up of 23.9 months, 30 patients (54%) demonstrated a clinical benefit of more than 1.3-fold enhanced progression-free survival compared with their previous therapy. Twelve patients (40% of responders) experienced exceptional responses lasting three times longer than expected for their respective disease. We conclude that therapy matching by scFPM is clinically feasible and effective in advanced aggressive hematologic cancers. Significance: This is the first precision medicine trial using a functional assay to instruct n-of-one therapies in oncology. It illustrates that for patients lacking standard therapies, high-content assay-based scFPM can have a significant value in clinical therapy guidance based on functional dependencies of each patient's cancer. See related commentary by Letai, p. 290. This article is highlighted in the In This Issue feature, p. 275

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBNAHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.

Background: Aggressive hematological malignancies in relapsed/refractory setting bear a dire prognosis with low cure rates and short survival. Matching these patients to therapies is challenged by complexity due to spatial and temporal tumor evolution and incomplete understanding of genotype to phenotype correlations. Direct functional testing could address these impediments. The EXALT trial is an interventional, one-arm study designed to assess the clinical value of next generation functional drug screening (ngFDS). An interim analysis on 17 patients suggested a clinical benefit (Snijder et al., Lancet Hematol. 2017). Methods: We applied image-based ngFDS to quantify differential ex-vivo sensitivity of primary patient tumor cells to respective non-tumor cells towards 136 small molecule drugs, including EMA approved for any indication or experimental. We screened bone marrow, peripheral blood or lymph node material from 143 patients who suffered from late stage aggressive hematological malignancies (acute leukemias, aggressive B- and T-cell lymphomas) , discussed the results in a multidisciplinary tumor board and recommended treatments to physicians (A). The primary endpoint of this study was the percentage of patients reaching a PFS-ratio (PFS(ngFDS treatment)/PFS(previous treatment)) of ≥1.3 with an H0 hypothesis < 15% patients. The secondary endpoint was overall response rate (ORR) defined as proportion of patients reaching complete remission (CR) or partial remission (PR). Additionally, we performed a post hoc analysis to evaluate the matching of ngFDS to drugs used in actual treatment (matching score of received treatment). Results: 56 (39%) patients were evaluable and treated according to ngFDS based recommendations. With 30 of 56 (54%) ngFDS guided patients experiencing a PFS ratio of ≥1.3, the primary study endpoint was reached. 11 patients (37%) had ongoing response at censoring date (B). The median follow-up was 718 days. The median number of days from sampling to treatment was 21 (range 4-77). The ngFDS treatment regimens consisted of a median of 2 drugs (range: 1-6). ORR was 55% for all evaluable ngFDS treated patients, 60% for the lymphoid subgroup and 41% for the myeloid subgroup. Patients on ngFDS guided treatment with performance status ECOG ≤ 1 had a median PFS of 207 days compared to a median PFS of 29 days for patients with higher ECOG (p < 0.001, C). 24 of 39 (62%) patients with ECOG ≤ 1 had a PFS ratio of ≥1.3 (D). In disease specific subgroup analysis median PFS of T-cell lymphoma patients was 235 days versus 60 days for B-cell lymphoma patients (p = 0.018, E). Age (≤60 vs. >60), sex, lineage (myeloid vs. lymphoid), number of previous treatment lines (≤2 vs. >2), and clinical presentation (leukemia vs. lymphoma) did not have an impact on PFS of ngFDS guided treatment. Post hoc analysis including additional 17 non-ngFDS treated patients demonstrated that only patients receiving treatment with a positive ngFDS matching score demonstrated clinical benefit (HR: 0.53, p=0.005; vs. HR: 1.4, p=0.4). ngFDS matched treatments resulted in higher PFS for patients with tumor samples that had a cancer cell fraction of 10-50% in comparison to patients with samples of lower or higher cancer cell percentage (HR:0.35, p=0.01). Conclusion: ngFDS could be integrated in the routine clinical work flow. ngFDS guided treatments led to high rates of PFS prolongation compared to previous treatments of individual patients. ngFDS guided treatment is feasible and effective in patients with late stage aggressive hematological malignancies. These results prompted a prospective randomized trial comparing treatment guidance based on ngFDS or comprehensive genomic profiling or physician's choice (EXALT-2 trial, NCT04470947). Figure Disclosures Vladimer: Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Jaeger:Karyopharm: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; True North: Honoraria, Research Funding; Miltenyi: Consultancy, Honoraria; CDR Life AG: Consultancy, Research Funding; F. Hoffmann-La Roche: Honoraria, Research Funding; Infinity: Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria. Krall:Allcyte GmbH: Current Employment, Current equity holder in private company, Other: Founder. Valent:Allcyte GmbH: Research Funding; Cellgene: Honoraria, Research Funding; Pfizer: Honoraria. Wolf:Celgene: Honoraria, Research Funding. Zielinski:MSD: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Imugene: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Merrimack: Consultancy, Honoraria, Speakers Bureau; Merck KGaA: Consultancy, Honoraria, Speakers Bureau; Fibrogen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Tesaro: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Shire: Consultancy, Honoraria, Speakers Bureau; Eli Lilly: Consultancy, Honoraria, Speakers Bureau; Athenex: Consultancy, Honoraria, Speakers Bureau. Superti-Furga:Allcyte GmbH: Current equity holder in private company, Other: Founder. Snijder:Allcyte GmbH: Current equity holder in private company, Other: Founder. Staber:Roche: Consultancy, Honoraria, Research Funding; Astra Zeneca: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria; msd: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.

Multiple myeloma (MM) is the second most common hematological malignancy and results from the clonal amplification of plasma cells. Despite recent advances in treatment, MM remains incurable with a median survival time of only 5-6years, thus necessitating further insights into MM biology and exploitation of novel therapeutic approaches. Both the ubiquitin proteasome system (UPS) and the PI3K/Akt/mTOR signaling pathways have been implicated in the pathogenesis, and treatment of MM and different lines of evidence suggest a close cross talk between these central cell-regulatory signaling networks. In this review, we outline the interplay between the UPS and mTOR pathways and discuss their implications for the pathophysiology and therapy of MM.

Introduction: Cereblon (CRBN) is the target for immunomodulatory drugs (IMiDs) such as thalidomide and its derivatives lenalidomide and pomalidomide, which are key therapeutics for hematologic malignancies such as multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). We have previously described a ubiquitin-independent chaperone-like function of CRBN, which stabilizes the transmembrane proteins CD147 and MCT1. IMiDs interfere with this chaperone-like function of CRBN in a competitive manner to mediate both their anti-tumor and their teratotoxic effects (Eichner et al. Nature Medicine 2016). So far, the underlying mechanisms of transmembrane protein maturation, the global impact of CRBN on the cell surface proteome and the precise molecular mechanism of IMiDs, especially their clinically well-established synergy with proteasomal inhibitors remain unclear. Methods: Novel CRBN-clients were identified by cross-validation of the CRBN-interactome with a cell surface proteomic screen. Various molecular and cell biological methods including immunofluorescence, flow cytometry, immunoprecipitations, GST-pulldowns, amino acid transport and proliferation assays were used to decipher underlying mechanisms. In vitro assays and in vivo xenograft experiments were performed using MM cell lines and patient-derived CD138+ MM cells. 18FDG- and 18FET-PET was used for imaging of xenografted tumors. Results: Our unbiased screening approaches imply a global role of CRBN in transmembrane protein maturation. In particular, we identify the amino acid transporter LAT1 and its functional subunit CD98hc as novel CRBN client proteins, which are frequently overexpressed in MM to drive cell proliferation. CD98hc/LAT1 become destabilized and inactivated upon IMiD treatment, which attenuates MM cell proliferation, tumor formation and perturbs the uptake of essential amino acids, thereby further linking IMiD-activity to tumor-metabolism. CD98hc/LAT1 destabilization only occurs in IMiD-sensitive patients and cell lines, thus being a potential biomarker to predict IMiD-response. Moreover, inhibition of LAT1 is cytotoxic in both IMiD-sensitive and -resistant cells, which makes it an attractive therapeutic option for IMiD-resistant and -refractory patients. Mechanistically, we show CRBN to function as a new selective co-chaperone of HSP90, which facilitates transmembrane protein maturation in a ubiquitin-independent way, which is impaired by IMiD-treatment. Conclusion: We establish CRBN as a transmembrane protein-specific co-chaperone for HSP90 and identify modulation of the CRBN-CD98hc/LAT1 axis as crucial means by which IMiDs mediate their anti-tumor activity. Notably, we specify CD98hc/LAT1 as valuable biomarkers for IMiD-response and druggable targets for IMiD-resistant and -refractory MM patients and beyond. Furthermore, this ubiquitin-independent mechanism solves the paradox regarding the well documented synergistic anti-myeloma activity of IMiDs and proteasomal inhibitors. Disclosures Götze: AbbVie: Membership on an entity's Board of Directors or advisory committees. Bassermann:Celgene: Consultancy, Research Funding.

Solute carriers (SLCs) are transmembrane proteins that transport various nutrients, metabolites, and drugs across cellular membranes. Despite the relevance of SLCs to cell homeostasis, metabolism, and disease states, for the majority of SLCs we lack experimental evidence regarding the nature of the cognate ligands, whether endobiotic or xenobiotic. Moreover, even for the roughly 20 SLCs for which inhibitors have been characterized, engagement assays in cells are limited to the accessibility of radiolabeled or fluorescent probes. The cellular thermal shift assay (CETSA) has been introduced as a powerful method to assess target engagement by monitoring ligand-induced changes in the thermal stability of cellular proteins. We addressed the question of whether CETSA could be modified to become routinely applicable to membrane transporters such as SLCs. We used SLC16A1 (MCT1) and SLC1A2 (EAAT2) as targets to establish robust conditions by which chemical engagement of SLCs can be detected. Using immunoblotting, we demonstrate that treatment with the SLC16A1 inhibitors AZD3965 and AR-C155858 stabilized endogenous SLC16A1 in HEK293 cell lysates as well as intact cells. In addition, the high-affinity ligand of SLC16A1, L-lactate, and the low-affinity ligand, formate, resulted in strong and weak stabilization of SLC16A1, respectively. Moreover, we observed stabilization of SLC1A2 upon treatment with the selective inhibitor WAY-213613. We propose that the experimental approach presented here should be generally and easily applicable for monitoring the engagement of chemical ligands by SLCs in cellular settings and thus assisting in their deorphanization.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.