Abstract
Multiple myeloma, a clonal plasma cell malignancy, has long provided a prototypic model to study regulatory interactions between malignant cells and their microenvironment. ...Myeloma-associated macrophages have historically received limited scrutiny, but recent work points to central and non-redundant roles in myeloma niche homeostasis. The evidence supports a paradigm of complex, dynamic and often mutable interactions between macrophages and other cellular constituents of the niche. We and others have shown that macrophages support myeloma cell growth, viability and drug resistance through both contact-mediated and non-contact-mediated mechanisms. These tumor-beneficial roles have evolved in opposition to, or in parallel with, intrinsic pro-inflammatory and tumoricidal properties. Thus, simple blockade of protective "don't eat me" signals on the surface of myeloma cells leads to macrophage-mediated myeloma cell killing. Macrophages also enhance the tumor-supportive role of mesenchymal stem/stromal cells (MSCs) in the niche: importantly, this interaction is bidirectional, producing a distinct state of macrophage polarization that we termed "MSC-educated macrophages." The intriguing pattern of cross-talk between macrophages, MSCs and tumor cells highlights the myeloma niche as a dynamic multi-cellular structure. Targeted reprogramming of these interactions harbors significant untapped therapeutic potential, particularly in the setting of minimal residual disease, the main obstacle toward a cure.
Summary
Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a ...proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase‐dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches. In myeloma tumour cells, MAP3K8 acts as mitogen‐induced MAP3K in mitosis and is required for TNFα‐mediated ERK activation. Pharmacological MAP3K8 inhibition results in dose‐dependent, tumour cell‐autonomous apoptosis despite contact with primary stroma. MAP3K8 blockade may disrupt crucial macrophage‐tumour cell interactions within myeloma niches.
Until now, ex vivo generation of CD34+ hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided ...invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow–derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14+ monocyte-derived macrophages to generate CD34+ cells from hESCs in vitro. Human ESC–derived CD34+ cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow–derived CD34+ cells. Finally, transplantation of purified hESC-derived CD34+ cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34+ cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.
Abstract 2444
Multiple myeloma genomes are characterized by complex structural and numerical abnormalities. Proteasome inhibitors are routinely used to treat multiple myeloma. Despite significant ...clinical success with these agents, development of resistance often limits therapeutic benefit. However, many questions remain unanswered regarding the molecular mechanisms underlying acquired resistance to proteasome inhibitors.
In order to understand the dynamics of structural evolution of the multiple myeloma genome under selective pressure afforded by proteasome inhibition and to identify targets to overcome acquired resistance, we derived global optical maps of two myeloma cancer genomes (DNA extracted from CD138+ tumor cells), obtained sequentially from the same patient before and after development of resistance to bortezomib, the prototypical therapeutic proteasome inhibitor. Optical Mapping offers a high throughput, single molecule, whole genome analysis that offers the highest rate of discovery of structural and numerical variants, free of the confounders associated with hybridization-based approaches. Briefly, the Optical Mapping System assembles entire genomes from large datasets of Rmaps (Rmap = a restriction-mapped individual genomic DNA molecule- see Figure 1) from which novel balanced and complex structural variants (2 kb – entire chromosomes) are discovered and tabulated by our pipeline (Figure 2).
We identified multiple structural variants including single nucleotide variations (SNVs), deletions, insertions, inversions, and loss of heterozygosity regions across the entire genome. Some of these variants are common to both bortezomib-sensitive and bortezomib-resistant genomes. We also discovered variants that were unique to the bortezomib resistant genome, implicating a role in acquisition of drug resistance. Many of these structural variants encompass genes, some of which have not been previously associated with multiple myeloma and bortezomib resistance, thus providing a rationale for further interrogation of these novel targets. In addition to novel potential targets, known recurrent events including del(13q) and a deletion spanning the CDKN2C/FAF1 locus on chromosome 1 were detected.
Future efforts are directed towards integration and correlation of optical maps with whole genome sequencing and transcriptional profiling as well as establishing the frequency of prioritized genomic perturbations in bortezomib-sensitive and –resistant patient populations. The integration of structural optical maps with base-pair sequence information and transcriptomic tracks will generate an entirely new view of the multiple myeloma cancer genome at a previously unseen resolution.
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No relevant conflicts of interest to declare.
Abstract Background aims For many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on ...this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed. Methods A combination of lineage-negative/c-kit-negative (Lin− /c-kit− ) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP+ ) adult CSC were co-cultured with GFP− cardiac-derived MSC Results MSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitro Conclusions MSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart.
Abstract Background aims In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important ...concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo –generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. Methods Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. Results The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. Discussion Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.
Abstract 443
Benefit from cytotoxic therapy in myeloma may be limited by persistence of residual tumor cells nested within favorable niches. However, the contribution of macrophages to the regulation ...of myeloma niches is still incompletely understood. We have previously shown that macrophages provide growth and anti-apoptotic signals to myeloma cells when grown in co-culture. These results prompted us to investigate the regulation of primary monocytes/macrophages that reside within myeloma niches in the bone marrow.
Unmanipulated CD14+ monocytic cells, freshly explanted from myeloma bone marrows, displayed a pre-dominantly pro-inflammatory transcriptomic profile when compared to normal monocytes. We found enhanced transcription of genes encoding pro-inflammatory mediators, known to support growth and survival of myeloma cells, such as TNFalpha, IL-1beta, IL-6, IL-8 and TACI. Downregulation of TGFbeta was also consistent with a pro-inflammatory (M1) signature. Interestingly, we also found concurrent transcription of some genes characteristic of “alternative macrophage activation” (M2 phenotype) such as IL-10 and IL1-receptor antagonist (IL-1RN). These results suggest that myeloma-associated macrophages, while being predominantly pro-inflammatory, display significant plasticity between the M1-M2 phenotypic extremes.
To obtain insights into the underlying mechanisms, we examined the role of TPL2 (Cot, MAP3K8), a serine/threonine kinase with central and non-redundant roles in regulating innate immune responses in macrophages following activation by Toll receptor (TLR) ligands and members of the TNF ligand superfamily. In myeloma-associated macrophages, we found constitutive activation of a TPL2 kinase-dependent, ERK-mediated pathway that promotes synthesis and processing of pro-inflammatory cytokines, including TNFalpha and IL-1beta. We also discovered constitutive activation of AKT at Ser473, a site dependent for its phosphorylation on TPL2 activity in macrophages responding to TLR signaling. Notably, the Akt/mTOR pathway limits the magnitude and duration of macrophage activation, in part through synthesis of the anti-inflammatory cytokine IL-10. These events involve signaling through STAT3. Accordingly, we discovered constitutive phosphorylation of STAT3 at a site regulated by TPL2 in activated macrophages.
In addition to non-tumor cell autonomous roles in regulating myeloma through macrophages, we showed a tumor cell-autonomous, growth-regulatory role of TPL2 kinase. Treatment of myeloma cells with a TPL2 small molecule inhibitor resulted in apoptosis that was not rescued by the presence of patient-derived stromal cells. We postulate that TPL2 inhibition interferes with growth signaling in myeloma cells because TPL2 has been shown to substitute for RAF proteins in growth signal transduction. Interestingly, we found that TPL2 was activated by phosphorylation as cells entered G2/M. Treatment with nocodazole increased the proportion of cells that co-expressed phosphorylated TPL2 and phosphorylated histone H3. Moreover, we found that TPL2 activity was required for MAPK pathway signal transduction in response to TNF receptor stimulation in myeloma cells.
Taken together, our results provide important novel insights into the regulation of macrophages within primary myeloma niches in the bone marrow. Plasticity between M1 and M2 phenotypes may correlate tightly with the actions of TPL2 kinase. In the myeloma niche, TPL2 activity helps to fine-tune macrophage activation by promoting synthesis and release of pro-inflammatory cytokines required by the myeloma tumor cell while engaging counter-regulatory mechanisms to prevent tissue destruction mediated by activated macrophages. Additionally, we described a growth regulatory role of TPL2 in the tumor cell itself. Thus, TPL2 blockade may disrupt crucial macrophage-tumor cell interactions within myeloma niches.
No relevant conflicts of interest to declare.
Abstract Background aims Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous ...hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. Methods We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4+ fraction of CD34+ HSCs, we could improve engraftment. Human cord blood-derived CD34+ cells and human bone marrow-derived MSCs were used for these studies. Results When MSCs were transplanted 1 week before CD34+ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34+ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4+ cells in the CD34+ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. Conclusions Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4–SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.
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