General MM

ASH 2017 | The role of the immune microenvironment in MM

The MM Hub team are delighted to be attending the 59th American Society of Hematology (ASH) Annual Meeting held in Atlanta, Georgia, from 9-12 December 2017. On Saturday 9 December, an oral session was held entitled: Session 651. Myeloma: Biology and Pathophysiology, excluding Therapy: New Insights into the Role of the Immune Microenvironment of Multiple Myeloma.The session was moderated by Qing Yi, from the Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, US and Karin Vanderkerken from the Free University of Brussels, Belgium, and the first three talks from this session are summarized in this article, which is based on data presented at the live session and therefore may supersede information in the pre-published ASH Abstract. 

Abstract 121: Low Dose Cyclophosphamide Potentiates the Anti-Myeloma Activity of Daratumumab via the Augmentation of Macrophage-mediated ADCP  

The first talk was delivered by Dr. Serika D. Naicker, from the Regenerative Medicine Institute, College of Medicine, Nursing and Health Sciences, National University of Ireland, Galway, Ireland and revealed data showing that low dose cyclophosphamide (Cy) acts in synergy with daratumumab.

The talk began with a description of the hallmarks of Multiple Myeloma (MM), and in particular how myeloma cells traffic through the vasculature to multiple sites in the bone marrow, leading to disease progression and poor prognosis. With the advancement of novel therapies, we now know that the immune system of a patient has an important role to play in treatment pathways -  immunocompetent patients respond better to chemotherapy than those that are immunocompromised, and consequently, there is a move towards more targeted therapy.

Daratumumab (dara), as one of these new targeted therapies, was then described. It was the first CD38-directed antibody developed specifically for the treatment of MM and is currently approved for the treatment of MM as a combo with a number of different reagents for Relapsed and Refractory (RR) MM patients. However, due to its limited side-effects and high level of efficacy, we are likely to see it move into frontline therapy for Newly Diagnosed (ND) MM patients, prior to transplant.

Interestingly, when dara is used as a combination therapy it is almost twice as effective as when it used as a monotherapy. However, despite obvious clinical synergies with other drugs the precise mechanism of action is still unknown. For example, dara is thought to act via both direct and indirect mechanisms: direct mechanisms including both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), as well as complement mediated cytotoxicity (CDC), leading to tumor cell death. In addition, dara leads to apoptosis via antibody-mediated cross-linking, and other indirect effects such as decreased immunosuppression and enzymatic inhibition. However, NK-cell mediated events such as ADCC are limited, as NK-cells appear to be depleted rapidly following dara treatment due to high levels of CD38 on NK-cells. Therefore, other immune cells must be involved in its efficacy.

In this study, experiments focussed on mechanisms surrounding macrophage (Mφ)-mediated ADCP, as they remain consistent in number throughout treatment. In addition, low dose cyclophosphamide (Cy) combined with antibody has been shown to alleviate the tumor cells’ ability to evade immune-mediated effects, and in particular immune-mediated ADCP. This was, therefore, the rationale for this study combining low dose Cy with dara, as it was postulated that low dose Cy with dara can induce changes in the BM microenvironment and potentiate dara to Mφ-mediated ADCP.

Key Data:
  • Myeloma cells in culture were treated with Cy (0-20 µM) for 24, 48 and 72 hrs and led to prolonged CD38 expression
  • Primary MM cells were collected from patients (pts) enrolled in the pCyBorD-Dara phase Ib clinical trial (NCT029518190): bone marrow (BM) and peripheral blood (PB) samples were collected prior to, and 24 hours after treatment and analyzed by flow cytometry
  • Marked reduction observed in CD47 expression (the so-called “don’t eat me” antigen) was observed on these cells after treatment with Cy
  • Transmigration assays revealed that Mφ exposed to tumor-conditioned medium (from MM cells exposed to Cy) showed an increase in chemotaxis markers (CCR5 and CCR2) and had a greater ability to migrate
  • Other changes observed in Mφs include:
    • Increase in CD64 expression (Fcgamma type I receptor); also confirmed with pt data
    • Decrease in PD-1 expression (checkpoint pathway), therefore offers another mechanism by which Cy can mediate ADCP
  • ADCP was inhibited when Mφs were exposed to cytochalasin-D (inhibitor of actin polymerization), further supporting a role for ADCP

In conclusion, Cy can mediate the resistance to treatment by enhancing levels of CD38 and indicates both phenotypic and functional changes. These observations confirm previous findings that suggest a role for Cy-directed Mφ mediated ADCP to enhance the efficacy of dara, and provided the rationale for further evaluation of a CyBorDara regimen in the CyBorD-Dara phase Ib clinical trial (NCT029518190) – see poster 3163 for patient outcomes.

Following the talk, it was asked if there was an opportunity for a metronome approach using very low doses of Cy. Dr. Naicker replied that in their study it is administered every few weeks, 24 hrs before dara, then along with the induction, consolidation, and maintenance. She said they could consider giving on a daily basis, but for the moment, in order to limit AEs and to simply drive the effect on the microenvironment, they had chosen this scheme. She was also asked if they had observed any changes in complement inhibitory proteins and replied that this work was ongoing. It was commented that daily doses of Cy should be considered and that there were certainly other mechanisms involved. Finally, it was asked if they had thought about using the metabolized form of Cy, to which Dr. Naicker replied that this is not commonly used in clinical practice.

Abstract 122: New Bone Cell Type Identified As Driver of Drug Resistance in Multiple Myeloma: The Bone Marrow Adipocyte (BMA)

The second talk was delivered by Dr. Carolyne Falank, from The Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, University of Maine, Orono, ME, US. The talk began with a description of the BM microenvironment as a pro-myeloma niche, and an explanation of the rationale to suggest that BM adipocytes (BMAs) drive MM survival signals in this niche, since obesity and aging are both risk factors for MM and correlate with higher adipose tissue.

The aim of the study outlined was to investigate the role between MM and BM-specific adipose tissue (BMAT), with a focus on response to therapy and with the hypothesis that BMAT contributes to drug resistance, via both soluble mediators and cell to cell adhesion.

BMAs were generated from either murine BM-mesenchymal stem cells (MSCs) to create mMAT or human MSCs to generate hMAT, and cultures of cells seeded onto scaffolds (tissue-engineered (TE) 3-D silk) scaffolds were examined. The cells were then cultured with luciferase-labeled MM cells, with or without dexamethasone (dex) and bortezomib (bort), and adiponectin. In addition, cell lines (OPM2 and MM1S) were also evaluated for adhesion, proliferation and drug resistance.

Key Data:
  • Dex treatment of MM1S cells leads to cell death; conditioned medium of BMAT rescues cells, also decrease in drug-induced apoptosis
  • MM cells up-regulate IL-6 when cultured with BMT-CM and further elevate IL-6 after dex treatment; IL-6 inhibitor led to decreased survival
  • IL-6 produced by MM cells can lead to drug resistance
  • Rescue of cells (from bortezomib and dexamethasone treatment) was observed with both BMAT conditioned media and direct cell culture; again due to drug-induced apoptosis  
  • MM cells up-reg VCAM 1 (as assessed by QRT-PCR), in particular in response to drug treatment
  • Drug resistance is also observed in 3D TE-BMAT and myeloma co-cultured for two weeks (2D culture has a 3-day limit)
  • Myeloma cells co-cultured with BMAT acquire adipogenic phenotype, suggesting “adipomimicry”:
    • OPM2 cells cultured with BMT express adipogenic gene signature: FABP4 and PPARϒ
    • MM1S cells cultured with BMAT-CM show secretion of adipogenic proteins: adiponectin, Pref-1, Leptin and Resistin
  • Increase in FABP4 gene expression was observed in OPM2 cells when co-cultured with hBMAT (3 donors)

Finally, a working model was described to explain how BM adipose tissue contributed to drug-induced resistance. Soluble factors from adipocytes signal an increase in IL-6 production, which contributes to survival against therapy, and up-regulation of proteins such as VCAM1, which lead to adipomimicry. Dr. Falank eluded to ongoing studies to examine the relationship in the other direction between MM cells and the effect they have on adipose tissue, such as a decrease in adipogenic genes.

A question was asked regarding how they can differentiate between mesenchymal stem cells and stromal cells, and if they can clearly see differences in effects following their differentiation protocol. Dr. Falank replied that they have significant success with differentiation and can effectively validate the resulting cells, and are confident that the effects observed are from adipose tissue. She was asked if they ever try IMiDs on adipocytes, and Dr. Falank said that this is something they are currently looking at.

Abstract 123: Single Cell Resolution Profiling Defines the Innate and Adaptive Immune Repertoires Modulated By Daratumumab and IMiDs Treatment in Multiple Myeloma (MM) 

The next talk was given by Dr. Paola Neri, MD, from the Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, AB, Canada, who described experiments in which the BM microenvironment of patients treated with daratumumab and other IMiDs was examined, in order to better understand mechanisms of resistance. The study aimed to define at the single cell level the BM immune repertoire of dara sensitive and resistant patients, to identify biomarkers of resistance and sensitivity, and to define potential means to reinstate sensitivity to both dara and IMiDs. This study aimed to address the question: What changes do the regimen of pomalidomide (pom) plus dara make to each cell subtype?

Key Data:
  • Serial BM aspirates from 20 pts treated with dara + pom in the MM014 trial were taken: prior to initiation of therapy, at the time of disease response and at relapse
  • The cells were then sorted for expression of CD138 and unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq)
  • Dara and pom modify the immune cell composition (immunome) of the BM niche in sensitive cells by:
    • Depletion of CD38highFCGR3AhighNK cells
    • Expansion of cytotoxic CD8 T effectors and TE T cells
    • Depletion of FCGR3Ahigh/CD14low monocytes
    • Expansion of M1 inflammatory macrophages
    • Depletion of immunosuppressive MDSCs and mesenchymal stem cells and plasmacytoid dendritic cells
  • Immunome of post dara/pom treated patients: Sensitive vs Resistant: 
  Sensitive Resistant
T cells

Expanded TSCM/CM

Expanded TEM

Exhausted T cells LGpos CD28neg

LAG3 > TIGIT >PDCD1

Macrophage/Monocytic cells M1 Macrophages

M2 Macrophages

Increased MDSCs

Natural Killer Cells Cytotoxic NK cells Regulatory NK cells

This signature represents potential biomarkers of response and indicates that LAG3 targeting could be a means by which to overcome resistance. The talk was well received and the question was posed as to whether they have any protein expression data to compliment what was shown. Dr. Neri replied that other groups have this data, but that in her group they are also trying to look at functional studies.

References
  1. S. D. Naicker et al. Low Dose Cyclophosphamide Potentiates the Anti-Myeloma Activity of Daratumumab via the Augmentation of Macrophage-mediated ADCP. #Abstract 121. 59th ASH Annual Meeting and Exposition 2017, Atlanta, GA.
  2. C. Falank et al. New Bone Cell Type Identified As Driver of Drug Resistance in Multiple Myeloma: The Bone Marrow Adipocyte (BMA). #Abstract 122. 59th ASH Annual Meeting and Exposition 2017, Atlanta, GA.
  3. P. Neri et al. Single-Cell Resolution Profiling Defines the Innate and Adaptive Immune Repertoires Modulated By Daratumumab and IMiDs Treatment in Multiple Myeloma (MM). #Abstract 123. 59th ASH Annual Meeting and Exposition 2017, Atlanta, GA.
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