Mariateresa Fulciniti

Heterogeneity of genomic evolution and mutational profiles in multiple myeloma

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment.

Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)-hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated beta-catenin level while down-regulating nuclear factor-kappaB (NF-kappaB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox–5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.

Elevated IL-17 produced by Th17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma

Elevated cytokines in bone marrow (BM) micro-environment (interleukin-6 [IL-6], transforming growth factor-beta [TGF-beta], and IL-1beta) may play an important role in observed immune dysfunction in multiple myeloma (MM). As IL-6 and TGF-beta are important for the generation of T-helper 17 (T(H)17) cells, we evaluated and observed a significantly elevated baseline and induced frequency of T(h)17 cells in peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMMCs) from MM patients compared with healthy donors. We observed significant increase in levels of serum IL-17, IL-21, IL-22, and IL-23 in blood and BM in MM compared with healthy donors. We also observed that myeloma PBMCs after T(H)17 polarization significantly induced IL-1alpha, IL-13, IL-17, and IL-23 production compared with healthy donor PBMCs. We next observed that IL-17 promotes myeloma cell growth and colony formation via IL-17 receptor, adhesion to bone marrow stromal cells (BMSCs) as well as increased growth in vivo in murine xenograft model of human MM. Additionally, we have observed that combination of IL-17 and IL-22 significantly inhibited the production of T(H)1-mediated cytokines, including interferon-gamma (IFN-gamma), by healthy donor PBMCs. In conclusion, IL-17-producing T(h)17 cells play an important role in MM pathobiology and may be an important therapeutic target for anti-MM activity and to improve immune function.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma Cells In vitro and In vivo

Purpose: We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Results: Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G2-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. Conclusion: These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma

Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Targeted Disruption of the BCL9/β-Catenin Complex Inhibits Oncogenic Wnt Signaling

Blocking BCL9/β-catenin interaction with a stapled peptide inhibits Wnt-dependent transcription and suppresses growth and metastasis in colon cancer and multiple myeloma. Stapling Down Oncogenic Wnt Signaling The Wnt signaling pathway plays ancient and essential roles—it’s required for embryonic development in all animals and for key functions in adult tissues. Dysregulation of the pathway, however, underlies multiple human cancers. The development of Wnt pathway inhibitors has received considerable attention, but to be useful, such inhibitors must not disrupt vital pathway functions. To address this issue, Takada and colleagues now target an interaction between two Wnt pathway proteins, one of which (BCL9) is highly expressed in tumors but not in the cells of tumor origin. Wnt signaling ultimately increases nuclear levels of the transcriptional activator β-catenin, which promotes the expression of genes involved in cell survival and division. Certain coactivators, including BCL9, can form a complex with β-catenin and increase such gene expression. Takada et al. aimed to disrupt the BCL9–β-catenin interaction with a structured peptide mimicking the BCL9 binding interface. BCL9 binds to a site on β-catenin that differs from those of other binding partners; contact occurs via an α-helical domain of BCL9. The authors stabilized peptides representing that domain by using hydrocarbon stapling, in which chemical restraints reinforce the α-helical structure. These peptides, unlike the unmodified version, were taken up by cancer cells. Additionally, one stabilized α helix of BCL9 (SAH-BCL9) bound β-catenin, selectively dissociating BCL9/β-catenin complexes and inhibiting Wnt-dependent transcription. SAH-BCL9, but not a mutant control peptide, reduced the proliferation of Wnt-dependent colorectal cancer and multiple myeloma cell lines. (SAH-BCL9 did not affect cell lines that do not express BCL9 or depend on Wnt signaling.) Furthermore, in mouse xenograft models of Wnt-driven colon cancer and multiple myeloma, SAH-BCL9 suppressed tumor growth, invasion into nearby tissues, and metastasis, as well as local formation of new blood vessels, in an apparently nontoxic manner. Thus, targeting the BCL9–β-catenin interaction may represent a useful approach for treating Wnt-dependent cancers. Additional experiments will be required to further optimize the drug-like properties of SAH-BCL9. Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the resulting aberrant transcription has proved difficult because the pathway comprises large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction of β-catenin with B cell lymphoma 9 (BCL9), a co-activator for β-catenin–mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain 2. We developed a stabilized α helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based antitumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9–β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a prototype therapeutic agent for cancers driven by deregulated Wnt signaling.

PI3K/p110{delta} is a novel therapeutic target in multiple myeloma.

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.