Selinexor is a novel, first-in-class oral selective inhibitor of nuclear export which blocks Exportin 1 (XPO1), forcing the nuclear retention and activation of tumor suppressor proteins, ultimately ...causing apoptosis in cancer cells. Selinexor has been approved for the treatment of patients with penta-refractory multiple myeloma (MM) who have received at least 4 prior therapies. We previously reported the discovery of a novel transcriptional signature and therapeutic targets for therapy resistant MM by comprehensive single cell RNA-seq analysis (scRNA-seq) of plasma cells (PCs) in patients with primary refractory MM (PRMM) enrolled in the KYDAR clinical trial (NCT04065789, carfilzomib Lenalidomide dexamethasone daratumumab for PRMM Cohen YC, Nature Med, 2021).
Here we report scRNA-seq analysis of PCs from patients with advanced refractory MM (aRRMM) (n=21) enrolled in an ancillary sub-study of a prospective clinical trial (XPORT-MM-028, NCT04414475), treated with selinexor combined with dexamethasone (Xd, in penta-refractory MM n=7), or with bortezomib, dexamethasone (XVd, in triple-class refractory TCR MM n=9), 5 patients participated in the ancillary study only. Median age was 75 years (range: 60-87), 50% were male, median time since active MM diagnosis was 4.8 years (range: 1.3-11.1). All treated patients (N=16) had TCR MM and 7/16 treated patients had penta-refractory MM. Single cell clustering analysis showed a unique molecular signature for each myeloma patient's PCs (Fig 1A), while patient-level analyses revealed a distinct transcriptional signature in aRRMM compared with PRMM (Fig 1B). aRRMM was characterized by upregulation of several pathways, including heparin growth factor (HDGF), Rho-GTPases activator (ARHGEF2), H3.3 histone variant and Prothymosin Alpha (PTMA) (Fig 1C-D). PPIA expression, which we previously identified as a biomarker and synergistic target for carfilzomib resistance, was low among healthy donors' PCs, progressively increased along with malignant evolution of plasma cell dyscrasia, from newly diagnosed MM, through PRMM, and was the highest in aRRMM (Fig 1C). We observed strong down-regulation of CD38 likely a consequence of daratumumab treatment and relapse in earlier lines.
Finally, we discovered differential expression of several genes between patients with MM refractory to versus non-refractory (achieving at least partial response by IMWG criteria or a progression free survival greater than 4 months) to a selinexor-regimen (Fig 1E), including up-regulation of XPOT, a tRNA exportin, and KPNB1 a nucleocytoplasmic transporter (Fig 1F-G). Protein-protein interaction enrichment analyses revealed mRNA splicing and capping as well as nucleocytoplasmic transport as up-regulated modules in the refractory patients potentially serving as a resistance mechanism for blockade of XPO1 mediated nuclear export by selinexor.
In summary, our study defines a roadmap for combining single cell RNA-seq profiling with clinical trials to stratify patients according to their level of anti-MM drug resistance, to define new biomarkers for drug resistance that may support personalized therapeutic decisions and reveal potential novel targets.
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Cohen: Karyopharm: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; neopharm / promedico: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau. Bentur: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Stemer: AbbVie: Consultancy. Avivi: Novartis: Speakers Bureau; Kite, a Gilead Company: Speakers Bureau. Amit: Neogene therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; CELLINK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Maruho Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck KGaA: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche Immunology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karophram: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to ...identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.
•Tumor cells express transcriptional programs of both immature and mature stages of normal PC development.•Survival of patients with AL and MM is inferior when tumor cells express transcriptional programs of more immature normal PC stages.
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Background
The relative contributions of microglia and infiltrating monocyte‐derived macrophages (MDMs) to containing Alzheimer’s disease (AD) are not fully understood. In the 5xFAD animal model of ...amyloidosis, disease‐associated microglia (DAM) expressing the Triggering receptor expressed on myeloid cells 2 (TREM2), are found in close proximity to amyloid beta (Aβ) plaques. Deletion of TREM2 results in the absence of DAM and in an increased Aβ‐plaque load. However, the necessity of TREM2 and DAM for resolving AD pathology is still debatable.
Method
Here, we activated systemic immunity by blocking the programmed cell death protein 1 / ligand (PD‐1/PD‐L1) pathway in TREM2‐/‐ and TREM2+/+ 5xFAD mice, to decipher the roles of the different myeloid populations in mitigating AD pathology.
Result
We found that anti‐PD‐L1 treatment resulted in cognitive improvement in TREM2‐/‐ and TREM2+/+ 5xFAD mice. In addition, in both TREM2‐/‐5xFAD and TREM2+/+5xFAD, the treatment resulted in a reduction in water soluble‐Aβ, while reduction of insoluble‐Aβ was observed only in TREM2+/+5xFAD mice. Eliminating monocytes using anti‐CCR2 antibody fully abrogated the observed effects of anti‐PD‐L1 treatment in TREM‐/‐5xFAD mice, and partially eliminated the effects in the TREM2+/+5xFAD. Single‐cell RNA‐seq of myeloid cells isolated from TREM2‐/‐5xFAD brains revealed that MDMs express unique scavenger receptors, previously linked to soluble‐Aβ removal, such as Macrophage scavenger receptor 1 (MSR1).
Conclusion
Overall, our findings highlight a novel TREM2‐independent pathway by which cognitive improvement and removal of soluble‐Aβ are achieved in an amyloidosis model. Thus, our results support the potential of MDM‐harnessing immunotherapy in treating AD patients, irrespective of whether they carry a TREM2 mutation.
Substantial progress in the treatment of Multiple Myeloma (MM) extends survival for many patients (Pts), though most Pts eventually relapse and become therapy refractory. Patients with induction ...resistant multiple myeloma (IRMM), either primary refractory or early (≤18 months) relapse, have a particularly compromised survival. New treatment strategies and molecular biomarkers for patient stratification and effective clinical care are needed.
We previously reported outcomes of KYDAR (NCT04065789) single-arm prospective clinical trial, in which pts primary resistant to a bortezomib-based induction achieved high rates of durable responses when treated with carfilzomib/daratumumab/lenalidomide/dexamethasone (Cohen YC et al. Blood (2019) 134 (Suppl 1): 982). We applied comprehensive single cell RNA-seq analysis of plasma cells (PCs) obtained from longitudinal bone marrow aspirate samples, taken from KYDAR participants (n=34), compared to newly diagnosed MM Pts (n=15) and to healthy controls (n=11). We discovered a novel MM resistance signature differentially expressed between IRMM and newly diagnosed MM groups. This “gene module is enriched for several pathways that were perturbed in the IRMM Pts, including mitochondrial stress genes, the ER and UPR pathway, and the proteasome machinery. Furthermore, differential gene expression analysis between KYDAR responders and non-responders unveil potentially druggable escape mechanisms. These include upregulation of genes associated with immune regulation, proteasome, apoptotic and ER-stress pathways, e.g. Cyclophilin A (PPIA) creating an elaborated signature and potential target list of pathways and escape mechanisms from a highly potent quadruple therapy. This signature includes many novel genes which were not previously described in the context of MM (Fig 1A).
Here we report external validation of this novel resistance signature among 908 MM Pts in the MMRF CoMMpass dataset. We found that our genes signature expression follows a normal distribution with no apparent sub-populations in naïve patients, but when examining Pts after multiple relapses, we detected gradient increase in our signature with a clear bi-model distribution (Fig. 1B). The prevalence of high module-1 expression was 5% in newly diagnosed Pts vs 14% in Pts in 3rd or subsequent relapse (p<0.001). Survival analysis on MMRF “module 1 high” (module 1 score > 200) Pts (n = 68) compared with the rest of the population (n = 711) revealed a striking hazard-ratio of 3.9 (2.22 - 6.87) with p-value = 4.57x10-17 (Fig 1C). Module-1 was highly predictive of treatment outcome in KYDAR trials, beyond FISH cytogenetics (Fig 1D).
We hypothesized that PPIA may function as a protective resistance gene in MM malignant cells, by accelerating protein folding pathways and reducing stress associated to proteasome inhibitors. In order to test whether PPIA is merely a marker for highly resistant patients or has a causal role in MM resistance to proteasome inhibitors, we used Cyclosporine A (CsA), a known inhibitor of PPIA, in a series of in vitro experiments, to explore it's potential synergy with carfilzomib, a proteasome inhibitor, on RPMI-8226 and U266B MM cell lines, expressing high levels of PPIA. Using MTS proliferation assay, we found that the combined CsA and carfilzomib therapy was significantly more effective than carfilzomib alone. Apoptosis as measured by Propidium Iodide, DAPI and Annexin V FITC staining, was dramatically increased in the combination therapy setting compared to carfilzomib or CsA monotherapy (Fig 1E).
In summary, our study defines a roadmap for combining single cell RNA-seq profiling with clinical trials. We reveal and externally-validate a novel transcriptional signature for therapy resistance. We show inhibition of PPIA, a potential target identified, by CsA, overcomes relative resistance of MM cell lines to carfilzomib. We anticipate that such studies will significantly improve the ability to define mechanism of action of treatment, molecularly characterize the Pts that may benefit from the treatment, and reveal potential novel targets.
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Tadmor:AbbVie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Medison: Consultancy, Speakers Bureau; Neopharm: Consultancy, Speakers Bureau; 6. Novartis Israel Ltd., a company wholly owned by Novartis Pharma AG: Consultancy, Speakers Bureau.
Background
Patients (Pts) with induction resistant Myeloma, either primary refractory or those relapsing early after completing induction, have dismal prognosis. To elucidate clinical and molecular ...pathways of refractoriness, we designed a clinical trial to evaluate the safety and efficacy of a quadruple salvage regimen of Carfilzomib (CFZ)/ lenalidomide (LEN)/dexamethasone (DEX) / daratumumab (DARA) (KRD-D) in newly diagnosed MM (NDMM) Pts with primary or secondary induction resistance (KYDAR trial) . Using a high resolution molecular tool of massively-parallel single-cell RNA-sequencing (MARS-seq) calibrated for this trial, we aimed to identify gene expression signatures associated with clinical response and resistance.
Methods
Population included forty NDMM Pts treated with a bortezomib-containing induction regimen, who either: i) Failed to achieve a minimal response after 2 cycles or a partial response after 4 cycles (Group A), ii) Had early relapse within 18 months from starting of therapy (group B) AND were not candidates (at screening) for autologous transplant. Following informed consent, all Pts were enrolled to receive KRD-D treatment in eighteen 28-day (d) cycles (Cy) or until disease progression/unacceptable toxicity. CFZ: 56 mg/m2 IV days 1,8,15 (Cy 1-9), d 1,15 (Cy 10-18). LEN: 25 mg (Frail: 15mg) d1-21; Dex 40mg (Frail: 20mg) weekly; DARA: IV 16mg/Kg weekly (Cy 1-2), q14d (Cy 3-6), q28d. After 18 cycles, Pts continued to receive DARA/Len. We applied longitudinally our calibrated protocol for scRNA-seq of MM PC (Ledergor et al 2018) to 31 KYDAR Pts at different time points, and compared them to NDMM and to healthy age and sex matched controls. Pts with partial response or better (IMWG criteria) after 3 cycles were defined as "responders".
Primary efficacy endpoints were safety and tolerability. Secondary endpoints included overall response rate (ORR); progression free survival (PFS), and overall survival (OS).
Results
Forty Pts were enrolled across 13 medical centers in Israel. Data on 36 Pts with a cutoff date of June 2nd 2019 are presented and will be further updated at the meeting. Patients had highly aggressive MM characteristics (Table): 78% had intermediate/high risk FISH abnormalities, 39% had extramedullary disease, and 38% were frail. At a current median follow-up of 4 months (range 1-12m), 20 Pts are still under active therapy. Of the 16 Pts that discontinued therapy: 9 had progressed, 2 had an adverse event (AE), 1 died, and 3 discontinued due to other causes. All Pts had at least 1 AE, most were grade 1-2; There were 90 treatment emergent AE (TEAEs) and 18 regimen related grade 3-4 AEs. TEAEs occurring in ≥2 Pts included neutropenia (38%), thrombocytopenia (26%), infections (21%), anemia (18%), pneumonia (15%), diarrhea (9%) and rash (9%). Durable and deep responses were achieved (Fig. A). ORR was 91% (30/34): near CR-stringent CR 24% / very good partial response 47% / partial response 21% / stable disease 3% / progressive disease 6%. At median follow-up PFS is 85% and OS is 97%.
Applying MARS-seq, we profiled a total of 39,492 PC sampled from 11 control subjects, 12 newly diagnosed MM patients and 31 KYDAR Pts at different time points. We classified the different cell groups based on expression levels of the most variable genes and used the differentially expressed genes to annotate malignant PC types (Fig. C). We identified multiple genes and pathways that are differentially expressed between healthy controls, NDMM and induction-refractory KYDAR Pts (Fig D). Importantly comparing the responder and non-responder Pts, in the KYDAR trial we identify statistically significant genes for responding and non-responding group, including critical immune checkpoints and regulatory genes in the proteasome, and apoptotic pathways (Figure E).
Conclusions
KRD-D quadruple regimen was safe and well tolerated, and provided an effective salvage in a cohort of induction resistant MM Pts, with an ORR of 91%, with deep responses achieved in 71%, equivalent to 2nd line triplet regimens in non-selected MM. Single cell analysis showed a dramatic change in the PC transcriptome following therapy. Furthermore, differences in gene expression patterns between responders and non-responders unveil potentially druggable escape mechanisms used by the highly resistant tumors including immune checkpoints that may serve as perspective biomarkers and therapeutic targets.
Disclosures
Cohen: Janssen Â: Consultancy; Amgen Â: Consultancy, Research Funding; Neopharm Â: Consultancy; Takeda Â: Consultancy; Madison Â: Consultancy; VBL Therapeutics Â: Employment. Ledergor:Immunai: Consultancy.
In the version of this Article initially published, an electronic conversion error led to errors in the names of Michal Safran and Baoguo Li. Their names have been corrected in the online version of ...the article.
Microglia and monocyte-derived macrophages (MDM) are key players in dealing with Alzheimer's disease. In amyloidosis mouse models, activation of microglia was found to be TREM2 dependent. Here, using ...Trem2
5xFAD mice, we assessed whether MDM act via a TREM2-dependent pathway. We adopted a treatment protocol targeting the programmed cell death ligand-1 (PD-L1) immune checkpoint, previously shown to modify Alzheimer's disease via MDM involvement. Blockade of PD-L1 in Trem2
5xFAD mice resulted in cognitive improvement and reduced levels of water-soluble amyloid beta
with no effect on amyloid plaque burden. Single-cell RNA sequencing revealed that MDM, derived from both Trem2
and Trem2
5xFAD mouse brains, express a unique set of genes encoding scavenger receptors (for example, Mrc1, Msr1). Blockade of monocyte trafficking using anti-CCR2 antibody completely abrogated the cognitive improvement induced by anti-PD-L1 treatment in Trem2
5xFAD mice and similarly, but to a lesser extent, in Trem2
5xFAD mice. These results highlight a TREM2-independent, disease-modifying activity of MDM in an amyloidosis mouse model.
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