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727 A Novel Therapy-Resistance Transcriptional Signature Based on Single Cell Analysis in Kydar Clinical Trial

Program: Oral and Poster Abstracts
Type: Oral
Session: 653. Myeloma/Amyloidosis: Therapy, excluding Transplantation; Novel Approaches for Relapsed/Refractory Myeloma and Amyloidosis
Hematology Disease Topics & Pathways:
Clinically relevant
Monday, December 7, 2020: 2:15 PM

Yael C Cohen, MD1,2*, Mor Zada, MSc3*, Shuang-Yin Wang4*, Chamutal Bornstein, PhD5*, Eyal David6*, Adi Moshe, PhD7*, Baoguo Li, PhD5*, Shir Shlomi7*, Moshe Gatt, MD8, Noa Lavi, MD9*, Chezi Ganzel, MD10*, Efrat Lutwak, MD11*, Ory Rouvio, MD12*, Iuliana Vaxman, MD, BA13*, Oren Pasvolsky, MD14*, Mouna Ballan15*, Tamar Tadmor, MD16*, Anatoly Nemets17*, Osnat Jarchowcky Dolberg18*, Yevgeny Chober19*, Olga Shvetz, MD20*, Merav Leiba21*, Ofer Shpilberg, MD, MPH22*, Nagib Deli23*, Irit Avivi, MD2,24*, Assaf Weiner, PhD3* and Ido Amit, PhD3*

1Hematology Department, self, tel aviv, Israel
2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
3Weizmann Institute of Science, Rehovot, Israel
4Wetzmann Institute of Science, Rehovot, Israel
5Immunology, Weizmann Institute, Rehovot, Israel
6Weizmann INstitute, Rehovot, Israel
7Weizmann Institute, Rehovot, Israel
8Department of Hematology, Hadassah-Hebrew University Medical Center-, Jerusalem, Israel
9Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, Haifa, Israel
10Hematology, Shaare Zedek Medical Center, Jerusalem, Israel
11Department of Hematology, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel
12Soroka Medical center, Be'er She'va, Israel
13Bellinson, Hod Ha-Sharon, None, Israel
14Institute of Hematology, Davidoff Cancer Center, Rabin Medical Center, Petah Tikva, Israel
15hematology, Carmel Medical Center, Haifa, Israel
16Hematology Unit, Bnai Zion Medical Center, Haifa, Israel
17hematology, 12Barzilai Medical Center, Ashkelon, Israel
18Hematology Department, Meir Medical Center, Kfar Saba, Israel
19HaEmek Medical Center, Afula, Israel
20Hematology, Kaplan Medical Center,affiliated with Hadassah and Hebrew University Medical School, Rehovot, Israel
21Assuta University Hospital, Faculty of Health Science, Ben-Gurion University of the Negev, Beersheba, Israel
22Institute of Hematology/Clinic of Histiocytic Neoplasms, Assuta Medical Centers, Tel-Aviv, Israel
23hematology, Rebecca Sieff Hospital, Zefat, Israel
24Department of Hematology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

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.

Disclosures: 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.

*signifies non-member of ASH