Chesi, Marta

Abstract Myeloma remains a virtually incurable malignancy. The inevitable evolution of multidrug-resistant clones and widespread clonal heterogeneity limit the potential of traditional and novel therapies to eliminate minimal residual disease (MRD), a reliable harbinger of relapse. Here, we show potent anti-myeloma activity of macrophage-activating immunotherapy (αCD40+CpG) that resulted in prolongation of progression-free survival (PFS) and overall survival (OS) in an immunocompetent, preclinically validated, transplant-based model of multidrug-resistant, relapsed/refractory myeloma (t-Vκ*MYC). αCD40+CpG was effective in vivo in the absence of cytolytic natural killer, T, or B cells and resulted in expansion of M1-polarized (cytolytic/tumoricidal) macrophages in the bone marrow. Moreover, we show that concurrent loss/inhibition of Tpl2 kinase (Cot, Map3k8), a MAP3K that is recruited to activated CD40 complex and regulates macrophage activation/cytokine production, potentiated direct, ex vivo anti-myeloma tumoricidal activity of αCD40+CpG–activated macrophages, promoted production of antitumor cytokine IL12 in vitro and in vivo, and synergized with αCD40+CpG to further prolong PFS and OS in vivo. Our results support the combination of αCD40-based macrophage activation and TPL2 inhibition for myeloma immunotherapy. We propose that αCD40-mediated activation of innate antitumor immunity may be a promising approach to control/eradicate MRD following cytoreduction with traditional or novel anti-myeloma therapies. Cancer Immunol Res; 3(8); 881–90. ©2015 AACR.

Abstract Background: The AAA-ATPase p97/VCP facilitates the extraction and degradation of ubiquitinated proteins by converting chemical energy into mechanical force. p97 is closely involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. p97 has been increasingly linked to cancer: it showed elevated protein expression in tumors, it can mediate the degradation of proteins in cancer-relevant pathways and is required for orchestrating the ubiquitin-governed DNA-damage response. In this context, p97 inhibitors may have an advantage versus other protein homeostasis inhibitors and may be active in solid tumors where 26S proteasome inhibitors, bortezomib and carfilzomib, have shown poor efficacy. We report here p97 inhibition as a novel approach to exploit cancer cell addiction to protein homeostatic mechanisms. Results: We have discovered novel small molecule inhibitors of p97 ATPase activity with nanomolar enzymatic and cellular potency. In cellular models, treatment of cancer cells with our lead compound CB-5083 causes disruptions in specific p97 functions, including ubiquitin-dependent protein degradation, ERAD, endocytosis and autophagy. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in polyubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Sub-chronic oral administration of CB-5083 is generally well-tolerated with <10% body weight loss and results in potent tumor growth inhibition in several solid tumor xenograft models. This result is in marked contrast to proteasome inhibitors that are inactive in the same solid tumor models. In the Vk*MYC transgenic mouse model of multiple myeloma, CB-5083 treatment resulted in >50% reduction in M-spike. Additional efforts are focused on the development of translational assays to monitor p97 target engagement and antitumor efficacy in upcoming clinical trials of CB-5083. Conclusion: These data demonstrate that CB-5083 is a potent inhibitor of p97 that translates to tumor growth inhibition in multiple rodent models of human cancer. Furthermore, CB-5083 appears to exhibit greater potency over current proteasome inhibitors that further validate targeting p97 and protein homeostasis in the treatment of cancer. Citation Format: Ronan Le Moigne, Steve Wong, Ferdie Soriano, Eduardo Valle, Daniel J. Anderson, Stevan Djakovic, Mary-Kamala Menon, Bing Yao, Julie Rice, Jinhai Wang, Szerenke Kiss Von Soly, Brajesh Kumar, Marta Chesi, P. Leif Bergsagel, Han-Jie Zhou, David Wustrow, Mark Rolfe, F. Michael Yakes. CB-5083 is a novel first in class p97 inhibitor that disrupts cellular protein homeostasis and demonstrates anti-tumor activity in solid and hematological models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 951. doi:10.1158/1538-7445.AM2014-951

Abstract The numerous and diverse genetic lesions in multiple myeloma (MM) make it difficult to discern those genetic abnormalities that may be suitable therapeutic targets. We have observed that the MYC locus is frequently rearranged in MM (about half of both patients and cell lines), suggesting that MYC dysregulation is a common feature of this disease. To evaluate MYC suppression as a therapeutic strategy in MM, we treated human myeloma cell lines with CPI203, a potent and selective inhibitor of bromodomain and extra-terminal (BET) proteins. The bromodomain inhibitor CPI203 has potency and selectivity similar to that of JQ1, but has improved pharmacokinetics for in vivo studies. From our collection of MM cell lines we have identified sensitive and resistant cell lines and characterized their response to CPI203 in detail. A number of conclusions have emerged from this work. First, response correlates with MYC protein levels after 3 days of treatment. MYC levels are significantly suppressed in sensitive lines and much less affected in resistant lines, supporting the hypothesis that MYC suppression alone determines cell fate in MM cell lines. In sensitive cell lines treatment with CPI203 caused a rapid (within 90 min.) decrease in MYC transcription that is consistent with the response being an intrinsic property of the cell lines. Inhibition of MYC transcription was followed by eventual cell cycle arrest and limited apoptosis in these sensitive cell lines. Both sensitive and resistant cell lines exhibited induction of Histone2H2BE gene in response to CPI203 treatment, indicating that drug efflux pumps alone do not mediate resistance. Surprisingly, several of the resistant cell lines exhibited an initial phase of sensitivity to BET inhibition. Upon drug addition, MYC transcription and MYC protein levels are repressed 2- to 5-fold, followed by a gradual recovery of MYC message and protein levels to the initial levels. In this second phase the cells are truly insensitive to BET inhibition: the drug remains active during this incubation and addition of fresh medium with drug does not affect MYC transcription. Furthermore, many aspects of the BET inhibition gene expression profile remain constant throughout this transition between sensitivity and resistance. These results suggest that the recovery phase is not a simple reversal of BET inhibition but rather the emergence of an alternative-signaling pathway that activates MYC expression in the presence of BET inhibition. Presently we are analyzing gene expression data to identify genes and signaling pathways responsible for this “induced resistance”. The identification of such pathways will help us better understand the molecular basis of the response to BET inhibition, which in turn might enable us to identify predictive biomarkers of the therapeutic response. Citation Format: Daniel L. Riggs, Marta Chesi, P Leif Bergsagel. Targeting MYC in multiple myeloma by BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2014-5489

Abstract Early-stage myeloma tumor cells are critically dependent on paracrine cytokine support originating from their bone marrow microenvironment whereas advanced-stage myeloma tumor cells may elaborate autocrine cytokine production mechanisms and/or cell-autonomous mutations in critical downstream signaling pathways (e.g., NFκB pathway). However, the molecular mechanisms underpinning the paracrine network in myeloma are unclear. The pro-inflammatory cytokine IL-1β has emerged as a major link between inflammation and cancer and has been validated as a therapeutic target in high-risk monoclonal gammopathy, the precursor to myeloma, as well as early-stage myeloma. To determine the source of IL-1β production in early-stage myeloma we analyzed paired purified cell fractions obtained from each of 5 patients at diagnosis: CD138+ tumor cells, CD14+ bone marrow-resident monocyte/macrophages as well as bone marrow-derived stromal/mesenchymal cells (BM-MSC). We found that in all cases, monocytes/macrophages were the predominant IL-1β synthesizer cell type in the myeloma microenvironment. Cytokine production by activated macrophages is controlled by the MAP3 kinase, TPL2 (Cot, MAP3K8). We have previously detected constitutive activation of TPL2-regulated signaling pathways in human myeloma-associated macrophages but its precise functional consequences have been unclear. To dissect the relevant mechanisms, we ablated Tpl2 in the genetically-engineered myeloma in vivo model, Vκ*MYC. Vκ*MYC animals activate MYC sporadically in B lymphocytes participating in germinal center reactions and develop a disease analogous to human multiple myeloma with production of paraprotein (monoclonal immunoglobulin), plasma cell infiltration of the bone marrow as well as end-organ damage (“myeloma kidney”, osteolytic lesions). Vκ*MYC+/Tpl2-null animals developed myeloma with a significantly prolonged latency and the disease burden was lower at all timepoints tested compared to controls. Analysis of monocytic cells from myeloma lesions showed that loss of Tpl2 did not result in macrophage repolarization to an unopposed M1 (tumoricidal/cytotoxic) phenotype. Instead, Tpl2-null myeloma-associated monocytes/macrophages exhibited severe defects in production of inflammatory cytokines, predominantly IL-1β, but also IL-6. Tpl2 loss did not have discernible impacts on tumor cell-autonomous growth and survival. Our results suggest that monocytes/macrophages and TPL2 kinase activity play a central role in orchestrating the inflammatory milieu of the myeloma niche. Disruption of the myeloma cytokine network through pharmacologic TPL2 kinase inhibition could provide novel therapeutic opportunity by interfering with the co-ordinate regulation of key pro-myeloma inflammatory cytokines through a targeted approach. Citation Format: Chelsea Hope, Samuel J. Ollar, Erika Heninger, Jeffrey L. Jensen, Ellen Hebron, Jaehyup Kim, Ioanna Maroulakou, Shigeki Miyamoto, Natalie Callander, Peiman Hematti, Marta Chesi, P. Leif Bergsagel, Fotis Asimakopoulos. TPL2 kinase regulates the inflammatory milieu of the myeloma niche. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2014-1169

Abstract The gene KDM6A/UTX was recently identified as a putative tumor suppressor (van Haaften et al). The gene is frequently lost in a variety of malignancies but most commonly in esophageal squamous cell carcinomas (8%) and multiple myeloma (10%). The initial characterization of UTX abnormalities in multiple myeloma utilized a mixture of 58 cell lines and patient samples. To establish the frequency of UTX inactivation in multiple myeloma we studied the copy number profiling dataset (n=239) available from the Multiple Myeloma Genomics Initiative (MMGI) and our internal dataset of 68 human myeloma cell lines which were characterized by array-based CGH (Agilent 400k), whole exome sequencing, and mRNA sequencing. We identified homozygous deletions in 2.5% of patients and 26.5% of cell lines. There are no mutations in the available whole genome and exome sequencing data from 38 patients in the MMGI that partially overlaps the 239 copy number samples sequencing. However, in the cell lines another 14.3% of samples showed loss of UTX through a variety of mechanisms (missense, nonsense, frameshifts, single exon deletions and insertions of LINE elements) detected by exome and mRNA sequencing, bringing the total percentage of cell lines with UTX inactivation to 40.8%. The increased frequency of UTX inactivation in cell lines likely suggests that it is associated with a more aggressive disease or promotes the growth of the tumor outside of the normal bone marrow microenvironment. As part of the cell line characterization phase of the project we found that ARD has a homozygous deletion encompassing the entire UTX locus while its isogenic counterparts, ARP-1 and CAG, express wild-type UTX at a level comparable to other cell lines without UTX abnormalities. To ensure these isogenic cell lines represent the same tumor not different tumors from the same individual we cloned the immunoglobulin receptor rearrangements in all there lines, which confirmed these are derived from the same original tumor. To develop this model system we first generated single cell clones from each of the three cell lines. Six clones from each were randomly selected and their DNA content was determined by flow cytometry. In all cases the clones matched the parental population in ploidy (ARD 3.2N, ARP 3.3N, CAG 3.8N). We randomly select one subclone from each cell line to characterize in detail using spectral karyotyping, array-based CGH, whole exome sequencing, mRNA sequencing, and CHIP-seq for H3K4me3, H3K27ac, H3K27me1, H3K27me2, and H3K27me3. The detailed characterization of this isogenic model system will make it an excellent platform for future work aimed at understanding how UTX loss contributes to myelomagenesis. Citation Format: Kristi Allen, Venkata Yellapantula, Quan Zhao, David Edwards, Marta Chesi, Leif Bergsagel, John Carpten, Jonathan Keats. Characterization of an isogenic model system for KDM6A/UTX loss in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5209. doi:10.1158/1538-7445.AM2013-5209

Abstract Malignant transformation is commonly associated with dysregulated ribosome biogenesis and for more than 100 years, pathologist have utilized the increase in size and number of nucleoli, the site of ribosome biogenesis as a marker for aggressive malignancies. Recently we demonstrated that hyperactivated RNA Polymerase I (Pol I) transcription, a rate limiting step in ribosome biogenesis, can be specifically targeted by the small molecule inhibitor, CX-5461. When evaluated for its anti-proliferative activity against a panel of genetically diverse human cancer cell lines, those derived from p53 wild type haemotological malignancies were the most sensitive (Drygin et al., Cancer Research, 2011). Evaluation of whether Pol I transcription inhibition could serve as therapeutic strategy in vivo revealed that CX-5461 administration significantly prolongs the overall survival of Eμ-Myc lymphoma bearing mice (Bywater et al., Cancer Cell, 2012). This survival advantage was associated with rapid activation of the ribosomal protein (Rp)-MDM2-p53 nucleolar stress response (Deisenroth et al., Oncogene, 2010) and subsequent induction of tumour-specific p53-dependent apoptosis (Bywater et al. Cancer Cell, 2012). New data and future directions: To further explore the therapeutic potential of Pol I transcription inhibition in haemotological cancers that are refractory to standard therapy, we employed mouse models of MLL-driven Acute Myeloid Leukemia (AML) (MLL/AF9 + Nras and MLL/ENL + Nras) (Zuber et al., Genes Dev, 2009) and V*κ-Myc-driven Multiple Myeloma (MM) (Chesi et al. Cancer Cell, 2009). CX-5461 administration significantly increased the overall survival time of AML-bearing mice (MLL/ENL Nras 17 days for vehicle vs 36 days for drug, P<0.0001; MLL/AF9 Nras 15 days for vehicle vs 23 days for drug, P 0.0009))without severe adverse effects. The extended survival of MLL-driven AML bearing mice was associated with acute activation of p53, an aberrant G2/M cell cycle progression and consequently a delay in tumour progression as monitored by bioluminescence-imaging. In contrast to CX-5461, treatment of AML -bearing mice with Cytarabine/Doxorubicin, a regime used as standard-therapy in the clinic failed to induce p53 in MLL-driven AML resulting in a significantly less survival time than for CX-5461. In ongoing experiments with MM-bearing mice, chronic CX-5461 administration robustly reduced the secretion of serum monoclonal Ig, as detected by serum protein electrophoresis (SPEP), and significantly prolonged survival time demonstrating that CX-5461 possess potent anti-tumour activity in this highly refractory MM model. Analysis of the molecular mechanism responsible for the therapeutic benefit of CX-5461 in MM is ongoing but appears to involve both p53 mediated apoptosis and aberrant cell cycle progression. Conclusion: These data demonstrate that hyperactivated Pol I transcription can be successfully targeted through small molecule inhibitors to therapeutically treat models of human AML and MM that are refractory to standard therapy.. Based on these and published results (Bywater et al., Cancer Cell 2012) and a favorable toxicology profile, we have launched a first-in-human clinical trial of this first in class drug, CX-5461 in patients with hematological malignancies. The outcomes of this trial will offer valuable insights as to whether inhibition of ribosome synthesis might offer a new non-genotoxic therapy approach in the fight against cancers, which are highly refractory to standard-therapies. Citation Format: Nadine Hein, Denis Drygin, Sean O'Brien, Carleen Cullinane, Geoff Matthews, Marta Chesi, Leif Bergsagel, Johannes Zuber, Scott Lowe, Ricky Johnstone, Rick Pearson, Grant McArthur, Ross Hannan. Inhibition of RNA Polymerase I transcription by CX-5461 as a therapeutic strategy for the cancer-specific activation of p53 in highly refractory haematological malignancies. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B16.

Abstract Multiple myeloma (MM) is a neoplastic plasma cell (PC) disorder of the oldster, and it is characterized by clonal proliferation of PC in the bone marrow (BM), monoclonal protein in biological fluids and organ dysfunction. While monoclonal gammopathy of undetermined significance (MGUS) often anticipates MM, the role of the cellular components of the BM microenvironment in the shift from MGUS to MM is unknown. Our aim was to define if the immune system has a role in this process. Thus, clinical signs of disease development and progression were investigated in the peripheral blood, bone marrow, kidney and bone of aging Vk*MYC mice to pinpoint the shift from MGUS to MM. We have also defined by immunohistochemistry and multiparametric flow-cytometry the phenotypic and functional characteristics of tumor infiltrating immune cells in the different phases of disease. We have found that the BM of Vk*MYC mice is the primary organ of PC accumulation, and accrual of PC in the BM associates with the presence of a measurable M-spike. Based on the frequency of PC in the BM and the quantification of M-spike in serum we were able to segregate MGUS from MM. The transition from MGUS to MM in Vk*MYC mice was characterized by increased vascularization of the BM that associated with local enrichment of proangiogenic molecules such as VEGF-A and IL18. The BM was also enriched in molecules that favor survival and proliferation of myeloma cells, and signs of osteoporosis accompanied these alterations. A comparison between Vk*MYC mice affected by MGUS and age matched wild type C57BL/6 mice revealed precocious alteration of the CD4/CD8 T cell ratio, increased percentage of IL2 and IL4 producing CD4+ T cells, and CD8+ T cells more prone to produce IFNγ in the BM of the former. In summary, our data suggest that early in disease development, yet unknown factors elicit an inflammatory reaction that significantly modifies the cellular composition of the BM and associates with neoangiogenesis and PC accumulation. Being the pathology in the Vk*MYC mouse similar to the human disease, our data might be relevant to understand the mechanisms of progression from MGUS to MM in humans. Citation Format: Arianna Calcinotto, Matteo Grioni, Maria Teresa Sabrina Bertilaccio, Marta Chesi, Leif Bergsagel, Giulia Casorati, Paolo Dellabona, Matteo Bellone. Modifications of the bone marrow microenvironment in the transition from monoclonal gammopathy of undetermined significance to multiple myeloma in Vk*MYC mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A83.

Key PointsCD28 delivers a pro-survival signal to MM cells via regulation of PI3K/Akt, FoxO3a, and Bim. Blockade of CD28:CD80/CD86 in vivo resensitizes MM cells to chemotherapy and significantly reduces tumor burden.

Key PointsWe describe a novel, druggable pathway that controls myeloma growth through macrophages in the myeloma microenvironment. Macrophages are dominant orchestrators of the inflammatory milieu of the myeloma niche.

Easy M Our immune systems can produce a vastly diverse repertoire of antibody molecules that each recognize and bind to a specific foreign antigen via a hypervariable region. However, there are a few bacterial antigens—such as Protein A, Protein G, and Protein L—that instead bind to the antibody's conserved regions and can bind to a large number of different antibodies. These high-affinity broad-spectrum antibody-binding properties have been widely exploited both in the laboratory and in industry for purifying, immobilizing, and detecting antibodies. Grover et al. (p. 656 ) have now identified Protein M found on the surface of human mycoplasma, which displays even broader antibody-binding specificity. The crystal structure of Protein M revealed how Protein-M binding blocks the antibody's antigen binding site. This mechanism may be exploited by mycoplasma to escape the humoral immune response.