PGC
            1β regulates multiple myeloma tumor growth through
            LDHA
            ‐mediated glycolytic metabolism

Key Points HERC4 is the first identified ubiquitin ligase that mediates c-Maf ubiquitination and degradation. HERC4 suppresses MM cell proliferation and delays MM tumor growth.

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Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽

10.1182/blood-2019-126298 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1844-1844

Author(s):

John Richards ◽

Myriam N Bouchlaka ◽

Robyn J Puro ◽

Ben J Capoccia ◽

Ronald R Hiebsch ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Tumor Cells ◽

Tumor Growth Inhibition ◽

Employment Equity ◽

Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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TLR4 signaling drives mesenchymal stromal cells commitment to promote tumor microenvironment transformation in multiple myeloma

Cell Death and Disease ◽

10.1038/s41419-019-1959-5 ◽

2019 ◽

Vol 10 (10) ◽

Cited By ~ 7

Author(s):

Cesarina Giallongo ◽

Daniele Tibullo ◽

Giuseppina Camiolo ◽

Nunziatina L. Parrinello ◽

Alessandra Romano ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Microenvironment ◽

Tumor Growth ◽

Cancer Progression ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Immune Escape ◽

In Vivo Model ◽

Th2 Response ◽

Tlr4 Signaling

Abstract Inflammation represents a key feature and hallmark of tumor microenvironment playing a major role in the interaction with mesenchymal stromal cells (MSC) in cancer progression. The aim of the present study was to investigate the crosstalk between MSCs and myeloma cells (MM) in the pro-inflammatory microenvironment promoting immune evasion and tumor growth. MSC were collected from patients with diagnosis of MGUS (n = 10), smoldering myeloma (n = 7), multiple myeloma at diagnosis (n = 16), relapse (n = 5) or refractory (n = 3), and from age-matched healthy controls (HC, n = 10) and cultured with peripheral blood mononucleated cells (PBMC) from healthy volunteer donors. Similarly to MM, we showed that MSC from smoldering multiple myeloma (SMM) patients activated neutrophils and conferred an immunosuppressive and pro-angiogenic phenotype. Furthermore, co-cultures of plasma cells (PC) and HC-MSC suggested that such activation is driven by MM cells through the switching into a pro-inflammatory phenotype mediated by toll-like receptor 4 (TLR4). These results were further confirmed using a zebrafish as an immunocompetent in vivo model, showing the role of MM–MSC in supporting PCs engraftment and Th2 response. Such effect was abolished following inhibition of TLR4 signaling in MM–MSC before co-injection with PC. Moreover, the addition of a TLR4 inhibitor in the co-culture of HC-MSC with MM cells prevented the activation of the pro-tumor activity in PC-educated MSC. In conclusion, our study provides evidence that TLR4 signaling plays a key role in MSC transformation by inducing a pro-tumor phenotype associated with a permissive microenvironment allowing immune escape and tumor growth.

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Role of microRNAs in Diagnosis, Prognosis and Management of Multiple Myeloma

International Journal of Molecular Sciences ◽

10.3390/ijms21207539 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7539

Author(s):

Amro M. Soliman ◽

Teoh Seong Lin ◽

Pasuk Mahakkanukrauh ◽

Srijit Das

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Growth ◽

Malignant Transformation ◽

Bone Disease ◽

Clinical Management ◽

Potential Role ◽

Plasma Cells ◽

The Usa

Multiple myeloma (MM) is a cancerous bone disease characterized by malignant transformation of plasma cells in the bone marrow. MM is considered to be the second most common blood malignancy, with 20,000 new cases reported every year in the USA. Extensive research is currently enduring to validate diagnostic and therapeutic means to manage MM. microRNAs (miRNAs) were shown to be dysregulated in MM cases and to have a potential role in either progression or suppression of MM. Therefore, researchers investigated miRNAs levels in MM plasma cells and created tools to test their impact on tumor growth. In the present review, we discuss the most recently discovered miRNAs and their regulation in MM. Furthermore, we emphasized utilizing miRNAs as potential targets in the diagnosis, prognosis and treatment of MM, which can be useful for future clinical management.

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Biallelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells

Cancer Science ◽

10.1111/cas.14386 ◽

2020 ◽

Vol 111 (5) ◽

pp. 1663-1675 ◽

Cited By ~ 1

Author(s):

Jo Kanasugi ◽

Ichiro Hanamura ◽

Akinobu Ota ◽

Sivasundaram Karnan ◽

Vu Quang Lam ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

Akt Signaling ◽

Myeloma Cells

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A Novel In Vivo Animal Model for Human Multiple Myeloma Based on Bioluminescence Imaging of Tumor Cell Growth.

Blood ◽

10.1182/blood.v106.11.3452.3452 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3452-3452

Author(s):

Anton C. Martens ◽

Henk Rozemuller ◽

Ellen van der Spek ◽

Lijnie Bogers-Boer ◽

Niels van de Donk ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Growth ◽

Quantitative Evaluation ◽

Bioluminescence Imaging ◽

Growth Curves ◽

Population Doubling ◽

Tumor Mass ◽

In Vivo Animal Model

Abstract Preclinical testing of new therapeutical strategies for the treatment of multiple myeloma (MM) requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. Models that meet both requirements have thus far not been described. Here we present a novel in vivo MM model by engraftment of MM U266 or RPMI-8226/S cells, both of human origin, into RAG2γc double knock-out mice. These mice are totally immune deficient because they lack T-, B and NK cells and the mice easily accept human cells (van Rijn et al., Blood 2003, Rozemuller et al., 2004). After intravenous injection of 2x106 MM cells engraftment and outgrowth occurred in all mice but was limited to the bone marrow compartment only. Flow cytometry (FCM) confirmed the presence of human CD45/38/138 positive MM cells in femur, spine, tibia and sternum bone specimens. Infiltration into other organs was not observed. In a next step MM cells were stably transduced using a retroviral vector encoding both the Green Fluorescent Protein (GFP) and firefly Luciferase (fLuc) marker genes. Technical advances in recent years in optical imaging by Bioluminescence Imaging (BLI) techniques allow visualization and quantification of bioluminescent light by detecting photons that are transmitted through mammalian tissue. When luciferase converts the substrate luciferin, photons are emitted that can be registered by using sensitive CCCD cameras. The absolute number of photons that are produced correlates, in our application, with local tumor mass. Mice were injected i.v. with 2x106 GFP-fLuc transduced MM cells (U266 or RPMI8226/S) and imaged weekly using BLI. Within 2 weeks after injection significant BLI signals were detectable. Per mouse 5-10 foci showed luciferase activity, predominantly in the pelvic region, skull, limbs, sternum, ribs and the spine. This low frequency of engraftment is in line with earlier reports on RPMI8226/S (Mitsiades et al., Cancer Res 2003). At 9 weeks the first mice developed hind leg paralysis which could be attributed to tumor associated spinal lesions. After 12 weeks the last mouse was sacrificed. BLI revealed that the intensity of light production at the various sites of tumor growth within individual mice as well as between mice showed a similar increase. This reflects an increase in tumor mass. Quantitative analysis of subsequent BLI images allowed construction of tumor growth curves of the total tumor mass per mouse as well as for the individual foci of MM growth in individual mice. We typically observed exponential growth, with growth curves running parallel with an average population doubling time of approximately 4–5 days. The range in which tumor growth could be monitored (and as a consequence also the response to treatment) spans 3–4 decades. In contrast with previously reported murine models for human MM where -next to bone marrow homing- also extra-skeletal tumors were observed our model almost exclusively shows homing of MM cells to the BM and is therefore more consistent with the clinical manifestation in myeloma patients. The major advantage of the model is the option for quantitative evaluation of the effect of a given treatment on the tumorload. Currently we are studying the efficacy of newly developed geranyl-geranyl-transferase inhibitors (GGTI). In conclusion, we have developed a novel in vivo model to study the characteristics of homing and outgrowth of MM and for quantitative evaluation of the efficacy of the therapeutic intervention applied.

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Novel Patient Derived Multiple Myeloma Model Reflects Sensitivity Towards Anticancer Treatment in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v126.23.3004.3004 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3004-3004 ◽

Cited By ~ 1

Author(s):

Julia B. Schueler ◽

Dagmar Wider ◽

Kerstin Klingner ◽

Gabrielle Melanie Siegers ◽

Annette M May ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Tumor Growth ◽

Treatment Options ◽

Tumor Biology ◽

Pdx Model ◽

Anticancer Treatment ◽

Nog Mice ◽

Pdx Models

Abstract Background Appropriate animal models for hematological malignancies are highly attractive, because they allow the study of the tumor biology and underlying disease mechanisms. They also constitute a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. To validate our multiple myeloma patient (pt)-derived xenograft (MM PDX) model (Schueler et al, Expert Opin Biol Ther, 2013), we systematically analyzed a panel of MM PDX with regard to their sensitivity towards standard of care treatment and compared these data with the pts' clinical outcome. Methods Bone marrow (BM) cells of 11 MM pts were implanted intratibialy (i.t.) into 103 NOD/Shi-scid/IL-2Rγnull (NOG) mice (n= 6-18 / pt sample). Mice were treated according to pts' therapy with VCD (Bortezomib, Cyclophosphamide, Dexamethasone), or to evaluate additional treatment options with Rd (Lenalidomide, Dexamethasone). Tumor growth and antitumoral activity in mice were assessed in tumor-bearing mice and compared to untreated control mice as well as to pts' response. Tumor growth in the mouse model was monitored by whole-body fluorescence-based in-vivo-imaging (IVI) using CF750-labeled α-HLA ABC antibody before and during treatment as well as 24h after last treatment cycle as described (Schueler J. PLOSone 2013). Mock-injected animals served as negative controls. Engraftment of human MM cells in murine organs was confirmed by flow cytometry and patho-histological analyses (immunostaining) at the end of the study. Results The pt cohort included a typical MM clientele for referral centers, with a median age of 75 years (range 56-85), median BM infiltration of 80% (20-90), and high- and standard-risk cytogenetics in 5 and 6 pts, respectively. All pts had advanced disease with Durie&Salmon stage III and active/symptomatic MM. All pts received VCD after diagnosis and BM sampling. MM cell engraftment could reliably be determined from experimental day 10 on in all 11 MM PDX models, at all assessed sites, namely within the BM, spleen and peripheral blood (PB) of recipient mice. Individual pt samples displayed distinct tumor growth patterns in vivo. Fluorescence intensity of engrafted murine organs ranged from 2- to 15-fold compared to mock injected control mice. Mean IVI signals in BM of recipient mice were 10-fold higher as compared to spleen signals, qualifying the BM niche as the preferred homing localization of pts' MM cells. Of note, both injected and non-injected BM sites were infiltrated by MM cells 10 days after tumor cell injection. Engraftment of human MM cells in the respective murine organs was confirmed by flow cytometry (HLA ABC, CD138, CD38) and histology and verified MM engraftment via both methods, confirming prior reports (Schüler PLOSone 2013; Groen Blood 2012;120:e9-16, Overdijk MAbs. 2015;7:311-21). The murine engraftment capacity was independent of MM type, disease stage, BM infiltration and cytogenetics of the donor pt. VCD was applied to 9 different MM PDX models and induced partial remission (PR; defined as at least 50% reduction of murine tumor load in BM, spleen and/or PB) in 5 out of 9 tested MM PDX models, whereas 2 cases each showed stable disease (SD) or progression (PD). The response rates in the mouse avatars mirrored the clinical outcome of the respective MM pts in 8/9 cases; only one MM pt showed serological and clinical PR, whereas the corresponding mice displayed SD. Rd induced PR in 1 and PD in a second MM PDX model, underlining the feasibility of MM PDX for drug screening approaches. Conclusions Due to the complex tumor biology, murine models of MM are still challenging. Our data support the preclinical rationale to use i.t.-injected NOG mice, since they closely resemble clinical MM with respect to symptoms, disseminated disease sites and response to anticancer treatment. Possible applications for the MM mouse avatars include development of new anticancer drugs as well as definition of biomarker strategies and selection of treatment options for individual pts with relapsed/refractory MM. The data of our preclinical study may serve as a useful future strategy to guide treatment decisions in refractory pts. The suitability as a drug development tool will be additionally determined performing treatment experiments with novel agents, e.g. elotuzumab or daratumumab. Disclosures Schueler: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment.

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Novel In Vivo Model of Multiple Myeloma Bone Disease Using ß-Tricalcium Phosphate Artificial Bone Grafts

Blood ◽

10.1182/blood.v126.23.4200.4200 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4200-4200

Author(s):

Miroslav Koulnis ◽

Homare Eda ◽

Loredana Santo ◽

Ka Tat Siu ◽

Janani Ramachandran ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

Bone Graft ◽

Bone Disease ◽

Tricalcium Phosphate ◽

Research Funding ◽

Bone Graft Substitute ◽

Artificial Bone ◽

Fetal Bone

Abstract Model systems to study Multiple Myeloma (MM) related bone disease exist but have a number of limitations. Disseminated MM models have variable cell homing and do not precisely recapitulate the human microenvironment interactions with myeloma cells. Severe combined immunodeficiency (SCID) mice engrafted with human fetal bone (SCID-hu) have been used by us, and are able to recapitulate the human bone marrow microenvironment. The fetal bone chips are however difficult to obtain, and vary in size and shape, complicating inter-sample comparison. Similarly, the poly-ε-caprolactone polymeric scaffold, previously used to seed murine or human stromal compartment, may not correctly reproduce bone destruction and inhibition of osteogenesis by MM as seen in patients, making this model difficult to test therapies targeting the MM niche. β-tricalcium phosphate (β-TCP) is a biocompatible and biodegradable bone graft substitute that is uniform in structure and easily available, and may be a viable alternative to overcome SCID-hu difficulties in modeling MM bone disease. Here, we utilized β-TCP bone graft substitute to develop a novel in vivo MM model where β-TCP permits the development of the bone microenvironment, supports MM development, and is technically feasible and highly reproducible. Using this model, we aim to better understand the biology of the niche in MM by genetically modifying its components and by testing new niche-targeting therapies. Our initial results show that osteogenesis takes place in the β-TCP bone graft, and the implant is supportive of MM tumor growth. Inter-scapular subcutaneous implantation of β-TCP alone, or co-implantation with human-derived stromal cell line HS27A in immunocompromised recipients resulted in the expression of osteogenic markers Runx2, alkaline phosphatase (ALP), Col1A1, and Osteocalcin (OCN), as well as a marker of bone resorption. Further, implants supported the growth of human-derived MM1.S and murine 5TGM1 cells, as visualized directly in vivo by serial luciferase bioluminescence imaging (BLI) and by immunohistochemistry. Modifying the niche compartment in Cre/iDTR animals with MM disease is an exciting novel strategy to understand which niche component in vivo may be targeted to suppress MM development. Mouse strains with promoter-specific Cre recombinase that induces the expression of the diphtheria toxin (DT) receptor (iDTR) can be utilized to selectively ablate a cell population of interest in vivo, via intraperitoneal DT injection. Here, we first utilized OCN-Cre/iDTR mice to test the deletion of mature osteoblasts in β-TCP artificial bone graft post-implantation. Our data show a dose-dependent reduction in osteoblastic markers OCN, ALP, Runx2, Sclerostin, Osteoprotegerin and RANKL. Importantly, DT ablation of osteoblasts in the β-TCP implant resulted in a significantly increased 5TGM1 tumor growth, as judged by BLI and tumor weight. Our data show that the mature osteocalcin-positive niche population is protective against MM disease. Ongoing studies of the β-TCP mouse model will address the relative contribution of various osteogenic populations to the course of MM development in vivo, and test the efficacy of novel MM drugs. Disclosures Raje: BMS: Consultancy; Amgen: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Onyx: Consultancy; BMS: Consultancy; AstraZeneca: Research Funding; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Millenium: Consultancy; Eli Lilly: Research Funding; Novartis: Consultancy; Acetylon: Research Funding; Millenium: Consultancy; Novartis: Consultancy; Acetylon: Research Funding.

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Bortezomib-Induced Proinflammatory Macrophages Contribute to Multiple Myeloma Cell Aggressiveness

Blood ◽

10.1182/blood.v126.23.5367.5367 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5367-5367

Author(s):

Ofrat Beyar Katz ◽

Neta Ben-Tsedek ◽

Irit Avivi ◽

Dror Alishekevitz ◽

Michael Timaner ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Growth ◽

Host Response ◽

De Novo ◽

Imaging System ◽

Conflicts Of Interest ◽

Scid Mice ◽

Tail Vein ◽

Inflammatory Macrophages

Abstract Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. Previous studies revealed that chemotherapy induces pro-tumorigenic host-mediated effects which could explain tumor re-growth and metastasis(Gingis-Velitski, Loven et al. 2011, Katz, Shaked 2014). Here we show that plasma from bortezomib-treated mice significantly increases migration, viability and proliferation of human MM cells in vitro, compared to plasma from control untreated mice. Comparable results were demonstrated with plasma obtained from patients with MM treated with bortezomib. Additionally, bortezomib induces the mobilization of pro-angiogenic bone marrow cells. Mice treated with bortezomib and subsequently intravenously injected with MM cells succumb to MM aggressiveness earlier than mice treated with the vehicle control(Figure 1). We show that pro-inflammatory macrophages contribute to MM cell aggressiveness in response to bortezomib treatment, in part by secreting interleukin-16(IL-16). Blocking IL-16 in conditioned medium obtained from bortezomib-treated macrophages generated reduced viability of MM cells in vitro. Accordingly, co-inoculation of MM cells with pro-inflammatory macrophages from bortezomib-treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti-tumor activity of bortezomib, this drug can induce host-driven pro-tumorigenic effects that may promote MM aggressiveness. Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system. Figure 1. Host response to bortezomib promotes MM aggressiveness in mice. Eight week old CB.17 SCID mice were injected intravenously with 1mg/kg bortezomib or vehicle (veh). Four and 24 hours later mice were inoculated through the tail vein with 5x106 CAG-luciferase+ cells (n=6-7mice/group). Tumor growth and expansion was assessed by IVIS imaging system. Disclosures No relevant conflicts of interest to declare.

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