Citations

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

SummarySixty‐four transplant‐eligible patients with newly diagnosed multiple myeloma (NDMM) received carfilzomib (days 1, 2, 8, 9, 15, 16), 300 mg/m2 cyclophosphamide (days 1, 8, 15), 100 mg thalidomide (days 1–28) and 40 mg dexamethasone (days 1, 8, 15, 22) in 28‐day cycles (CYKLONE regimen). Carfilzomib was dose‐escalated to 15/20, 20/27, 20/36 and 20/45 mg/m2 to determine the maximum tolerated dose (MTD), which was 20/36 mg/m2. Regardless of attribution, common Grade 3 or higher adverse events were lymphopenia (38%), neutropenia (23%) and anaemia (20%). All peripheral neuropathy (31%) was Grade 1 and considered most likely to be thalidomide‐related. Common cardiac or pulmonary events of any grade in ≥5% of patients included dyspnoea (20%) and cough (6%). Overall (N = 64), 91% of patients achieved a best response of partial response or better across all cycles of treatment, including five patients with complete responses. At the MTD (n = 29), 59% of patients achieved a very good partial response or better after four cycles (primary end point). Stem cell collection was successful in all patients in whom it was attempted (n = 42). Progression‐free survival and overall survival at 24 months was 76% and 96%, respectively (median follow‐up of 17·5 months). CYKLONE appears highly efficacious in NDMM patients, with manageable toxicities.

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 PointsWe identified CRBN-binding proteins and confirmed recent work implicating the Ikaros transcription factors as important members. Low IZKF1 levels predict lack of IMiD responsiveness and shorter OS; IZKF1 expression is a potential predictive biomarker for clinic application.

The celebration of the 50th anniversary of the founding of the American Society of Clinical Oncology provides the occasion to review the progress that has been made in the biology and treatment of multiple myeloma. With the advent of melphalan and cyclophosphamide in the early 1960s the median survival of patients with multiple myeloma more than doubled from 10 months to approximately 24 months. Throughout multiple clinical trials in the 1970s and 1980s, melphalan and prednisone remained the gold standard, with a 3-year survival of 42%. The use of high-dose melphalan with autologous hematopoietic stem cell support provided an incremental advance in the 1990s. The outlook for patients was dramatically improved in the 2000s with the introduction of thalidomide analogs and proteasome inhibitors, so that the 3-year survival of patients treated in 2008 with melphalan and prednisone had increased to 66%. The 2010s are dominated by studying the optimal combination, sequence, and duration of therapies. These clinical advances have occurred along with our evolving understanding of the molecular pathogenesis of myeloma. Myeloma can be divided into two main groups: hyperdiploid, with multiple trisomies of odd-numbered chromosomes, and nonhyperdiploid, with recurrent immunoglobulin heavy chain gene translocations. Disease progression is associated with rearrangements of MYC, the most common mutation in myeloma, present in nearly half of patients. Genomic studies have highlighted marked subclonal heterogeneity that poses one of the main challenges to successful control of the disease. This problem will be addressed in future studies in the 2020s, which will include a focus on immunologic approaches such as monoclonal antibodies, checkpoint inhibitors, engineered T-cells, and novel immunomodulators.

Significance We show that DNA double-strand breaks (DSBs) can be repaired by insertion of 50- to 1,000-bp sequences termed “templated-sequence insertions” (TSIs) derived from distant regions of the genome. Additional experiments indicate that the source of template for repair was primarily nuclear RNA. This mode of DNA-DSB repair by insertion is not restricted to experimentally produced breaks but also occurs at the site of spontaneous DNA DSBs in human cells. These TSIs are polymorphic in the human genome, suggesting that some TSIs occur in germ cells or embryos. Recognition of these TSIs is important in interpreting structural variations in short-read sequencing studies and provides additional polymorphic markers for population and evolution studies. This error-prone form of DNA repair may play a role in genetic diseases.

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.