PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Modifying Graft-versus-Host Disease in a Humanized Mouse Model by Targeting Macrophages or B-Cells.

Humanized mouse models can well be modified to study specific aspects of Graft-versus-Host Disease (GvHD). This paper shows the results of both macrophage depletion and (early) B-cell depletion in a humanized mouse model using RAG2-/- γc-/- mice injected with HuPBMCs. Macrophage depletion showed a significant decrease in survival and also lead to a change in the histomorphology of the xenogeneic reaction. Higher levels of infiltrating B-cells were observed in various organs of mice depleted for macrophages. With (early) B-cell depletion using Rituximab, a clear improvement on clinical symptoms was observed, even when probably only inactivated B-cells were deleted. However, the histological examinations only showed a significant morphological effect on liver fibrosis. This may be related to a difference in the mRNA levels of TGF-ß. Also, lower mRNA levels of Tregs in some organs were observed after Rituximab treatment, which contradicts that a higher number of Tregs would always be related to less severe GvHD. Our data show that both macrophage depletion and (early) B-cell depletion in a xenogeneic mouse model can influence the clinical, histological, and cytokine production of a GvHD response.

Liposomal dexamethasone inhibits tumor growth in an advanced human-mouse hybrid model of multiple myeloma.

Glucocorticoids are the cornerstone in the clinic for treatment of hematological malignancies, including multiple myeloma. Nevertheless, poor pharmacokinetic properties of glucocorticoids require high and frequent dosing with the off-target adverse effects defining the maximum dose. Recently, nanomedicine formulations of glucocorticoids have been developed that improve the pharmacokinetic profile, limit adverse effects and improve solid tumor accumulation. Multiple myeloma is a hematological malignancy characterized by uncontrolled growth of plasma cells. These tumors initiate increased angiogenesis and microvessel density in the bone marrow, which might be exploited using nanomedicines, such as liposomes. Nano-sized particles can accumulate as a result of the increased vascular leakiness at the bone marrow tumor lesions. Pre-clinical screening of novel anti-myeloma therapeutics in vivo requires a suitable animal model that represents key features of the disease. In this study, we show that fluorescently labeled long circulating liposomes were found in plasma up to 24 h after injection in an advanced human-mouse hybrid model of multiple myeloma. Besides the organs involved in clearance, liposomes were also found to accumulate in tumor bearing human-bone scaffolds. The therapeutic efficacy of liposomal dexamethasone phosphate was evaluated in this model showing strong tumor growth inhibition while free drug being ineffective at an equivalent dose (4 mg/kg) regimen. The liposomal formulation slightly reduced total body weight of myeloma-bearing mice during the course of treatment, which appeared reversible when treatment was stopped. Liposomal dexamethasone could be further developed as monotherapy or could fit in with existing therapy regimens to improve therapeutic outcomes for multiple myeloma.

A Rational Strategy for Reducing On-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors by Affinity Optimization.

Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using "light-chain exchange" technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.

Establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches.

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies.

Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and ß-tricalcium phosphate.

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone-forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene-marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase-GFP-labelled MSCs were seeded on hydroxyapatite (HA) or ß-tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase-positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two-fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four-fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell-scaffold-based BTE.

Optimal selection of natural killer cells to kill myeloma: the role of HLA-E and NKG2A.

Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-E(low) expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2(-/-) γc(-/-) mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and NK cell degranulation upon co-culture with myeloma cells revealed that KIR-ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLA-class I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR-ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy.

Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma.

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.

Preclinical Evidence for the Therapeutic Potential of CD38-Targeted Immuno-Chemotherapy in Multiple Myeloma Patients Refractory to Lenalidomide and Bortezomib.

PURPOSE: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. EXPERIMENTAL DESIGN: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. RESULTS: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. CONCLUSIONS: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients.

Target antigen density governs the efficacy of anti-CD20-CD28-CD3 ζ chimeric antigen receptor-modified effector CD8+ T cells.

The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR-T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR-T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR-T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

Deazaneplanocin a is a promising drug to kill multiple myeloma cells in their niche.

Tumoral plasma cells has retained stemness features and in particular, a polycomb-silenced gene expression signature. Therefore, epigenetic therapy could be a mean to fight for multiple myeloma (MM), still an incurable pathology. Deazaneplanocin A (DZNep), a S-adenosyl-L-homocysteine hydrolase inhibitor, targets enhancer of zest homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2) and is capable to induce the death of cancer cells. We show here that, in some MM cell lines, DZNep induced both caspase-dependent and -independent apoptosis. However, the induction of cell death was not mediated through its effect on EZH2 and the trimethylation on lysine 27 of histone H3 (H3K27me3). DZNep likely acted through non-epigenetic mechanisms in myeloma cells. In vivo, in xenograft models, and in vitro DZNep showed potent antimyeloma activity alone or in combination with bortezomib. These preclinical data let us to envisage new therapeutic strategies for myeloma.

Differential effects of nitrostyrene derivatives on myelopoiesis involve regulation of C/EBPα and p38MAPK activity.

Bone marrow failure syndromes and MDS represent a heterogenous group of diseases, characterized by ineffective myelopoiesis, the risk of clonal evolution and a generally poor response to chemotherapy-based treatment regimen. Nitrostyrene derivatives have been studied as protein phosphatase inhibitors in various tumor models. Pharmacological studies have identified nitrostyrene as the structural core underlying a pro-apoptotic effect in tumor cells, yet their effects on normal cells, including those of the hematopoietic system, are largely unknown. In this study, utilizing umbilical cord blood-derived myeloid progenitor cells, patient-derived bone marrow cells, and a (BALB/c) mouse model; we investigated the effects of treatment with two nitrostyrene derivatives (NTS1 and NTS2) on myeloid development. We demonstrate that these compounds stimulate the expansion and differentiation of myeloid progenitors in vitro and improve myeloid reconstitution after chemotherapy-induced bone marrow depletion in vitro and in vivo. These effects were accompanied by increased C/EBPα expression and activity and inhibition of the p38MAPK signalling pathway. Together, our data suggest that nitrostyrenes improve myelopoiesis and represent potential new treatment strategies for patients suffering from bone marrow failure syndromes, hypocellular myelodysplastic syndrome and chemotherapy-induced aplasia.

Preclinical activity of the oral proteasome inhibitor MLN9708 in Myeloma bone disease.

PURPOSE: MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models. EXPERIMENTAL DESIGN: The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities. RESULTS: Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-κB ligand)-induced NF-κB activation, F-actin ring disruption, and diminished expression of αVß3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/ß-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow-disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). CONCLUSION: Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma.

Brief report: Autologous stem cell transplantation restores immune tolerance in experimental arthritis by renewal and modulation of the Teff cell compartment.

OBJECTIVE: Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). METHODS: For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). RESULTS: ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. CONCLUSION: Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells.

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing "bulk-cultured" BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.