Class of 2012 Abstract / Specific Aims: Autologous stem cell transplant (ASCT) is the current gold standard following induction therapy to improve survival of multiple myeloma (MM). Lenalidomide (LEN) is used for treatment of MM before ASCT, but exposure may impair autologous peripheral blood stem cell (PBSC) mobilization. Chemomobilization with cyclophosphamide (CTX) has not been evaluated in this setting. CTX + filgrastim was investigated to determine if LEN-associated mobilization impairment can be abrogated. Methods: 36 pts (group A=12 pts who received ≥2 cycles of LEN and group B=24 pts without LEN) were analyzed retrospectively. Baseline characteristics were matched (p>0.05 for all variables). All pts received CTX (median group B, 1.5g/m2; median group A, 3gm/m2(p=0.18)) and filgrastim 10μg/kg/day. Primary outcomes include number of CD34+ cells collected and number of leukapheresis sessions. Secondary outcomes include failure to collect CD34+ cells and total CD34+ cells collected after second leukapheresis. Main Results: Total median number of CD34+ cells collected in group B=9.15x106/kg CD34+ cells and group A=7.43x106/kg CD34+ cells (p=0.159). Median number of apheresis sessions in group B=2 and group A=3 (p=0.42). Two of 12 pts with antecedent LEN usage failed to collect while no patient without previous LEN exposure failed to collect (p=0.105). Total number of CD34+ cells collected after 2 apheresis sessions for group B=8.13x106/kg CD34+ cells and group A=3.34x106/kg CD34+ cells (p=0.06). Conclusions: Chemomobilization with CTX + filgrastim yields robust PBSC collections irrespective of antecedent lenalidomide. There was a trend towards lesser PBSC collection in LEN-treated pts.

The goal of this study is to determine whether SKI-606, a Src/abl inhibitor, potentiates chk1 inhibitor UCN-01 to induce apoptosis in multiple myeloma cells, and what mechanism maybe involved. We found that the co-exposure of human myeloma cells (e.g., U266, RPMI8226, MM.1S and its dexamethasone-resistant counterparts MM.1R) to minimally toxic concentration of SKI-606 (e.g., 1-2 ƒÝM) and UCN-01 (e.g., 100-150 nM) resulted in dramatic increase in mitochondrial damage and apoptosis. In our previous reports, it has been well demonstrated that activation of Ras/Raf/MEK/ERK pathway represents a critical cytoprotective response in cells exposed to UCN-01. Moreover, Src is required to activate this pathway by growth factors and cytokines. To this end, we examined whether the Src inhibitor enhances UCN-01 lethality through interruption of Ras/Raf/MEK/ERK signaling cascade. Firstly, it was found that co-adminstration of SKI-606 markedly diminished ERK phosphorylation/activation induced by UCN-01, accompanied by an increase in cdc2 activation. Furthermore, myeloma cells with ecpotic expression of either active mutant Ras (Q61L) or constitutive active MEK1 were significantly resistant to combined treatment with SKI-606 and UCN-01, indicating Src inhibition acts upstream of Ras/Raf/MEK to potentiate UCN-01 lethality. Conversely, stable expression of dominant-negative mutant Ras (S17N) markedly sensitized myeloma cells to this combination regimen. Lastly, ectopic expression of kinase inactive (K297R) or dominant-negative (K296R/Y528F) mutant Src blocked ERK activation by UCN-01 and thereby sensitized myeloma cells to UCN-01. Together, these findings indicate that Src inhibitors act through a Ras/Raf/MEK-dependent mechanism to prevent ERK activation in UCN-01-treated cells, resulting in the synergistic induction of cell death.

Previously, we have found that the co-administration of MEK1/2 inhibitors and Chk1 inhibitors synergistically induce multiple myeloma cell apoptosis through upregulation of the BH3-only pro-apoptotic protein Bim. However, these apoptotic events were largely blocked by the characteristic over-expression of Bcl-2 of Bcl-xL in multiple myeloma cells. HA14-1, a small molecule Bcl-2 inhibitor, may therefore circumvent this resistance to apoptosis by blocking Bcl-2 and Bcl-xL anti-apoptotic protein actions. In our project, we hypothesize that the co-administration of HA14-1 with MEK/Chk1 inhibitors will enhance apoptosis in multiple myeloma (MM) cells. To test this hypothesis, we exposed MM cells U266 and RPMI8226, or those cells with Bcl-2 over-expressing stable clones to minimally toxic concentrations of MEK1/2 inhibitor (PD184352) with Chk1 inhibitor (CEP3891) for 24 hours, followed by the Bcl-2 inhibitor (HA14-1). To date, our data indicates that co-administration of HA14-1 with the PD184352/CEP3891 regimen significantly enhances apoptotic death in U266/Bcl-2 multiple myeloma cells compared with the PD184352/CEP3891 regimen. Future studies are designed to elucidate mechanisms underlying Bcl-2 and Bcl-xL anti-apoptotic protein interactions with the Bak and Bim apoptotic proteins, focusing release of Bak and Bim from Bcl-2/Bcl-xL, and subsequent Bax/Bak activation.

Despite improved treatments for Non-Hodgkin’s Lymphoma (NHL) and Multiple Myeloma (MM), most patients eventually relapse and these diseases remain largely incurable. This has precipitated recent research into more clinically relevant in vitro models to enable development of more effective therapies. We have validated and standardised two in vitro primary culture models using tumour samples derived from patients with NHL, Chronic Lymphocytic Leukaemia (CLL) and MM. Several novel findings have been demonstrated. In vitro sensitivity of primary NHL cells cocultured in a CD40L model predicted clinical response to bortezomib in patients receiving the drug in a phase II trial. In vitro sensitivity correlated with CD40 expression, identifying a potential surrogate biomarker for response to bortezomib. The novel HDAC inhibitor, UCL67022 was 10-fold more potent than vorinostat in NHL and produced synergy when combined with bortezomib. UCL67022 maintained its potency in primary MM samples grown in an HS-5 stromal model. It modulated cytokine secretion resulting in downregulation of cytokine-induced signalling pathways (JAK/STAT3). A novel Hsp90 inhibitor, KW-2478 maintained activity in the HS-5 model and enhanced the activity of bortezomib and melphalan. Hsp70 was identified as a potential surrogate biomarker to monitor the combinatorial effect in future clinical trials. A highly synergistic and schedule-dependent cytotoxic effect occurred when primary MM cells were pre-treated with melphalan followed by bortezomib, with important implications for future clinical trial design. IL-6, IL-8 and VEGF levels correlated with resistance to bortezomib and melphalan and were associated with activation of JAK/STAT, MAPK and PI3K/Akt signalling pathways. Antibody neutralization of IL-6, IL-8 and VEGF resulted in restoration of drug sensitivity. We have therefore demonstrated the ability of primary culture models to predict response to chemotherapy, to identify therapeutically beneficial novel agents and to enable study of tumour microenvironmental interactions responsible for drug resistance in patients with haematological malignancies.

The unfolded protein response (UPR) is a complex signalling pathway activated in response to endoplasmic reticulum stress. In recent years, the UPR has been implicated in cancer and chemosensitivity, particularly in solid tumours. This thesis investigated the potential value of targeting the UPR as a novel therapeutic approach in haematological malignancies using a panel of cell lines representing AML, lymphoma and myeloma. The UPR was constitutively active in these haematological cancer cell lines, with differential activation of key UPR proteins both in the panel and between the panel, peripheral blood mononuclear cells and the colorectal cancer cell line HT-29. A number of strategies were used to modulate the UPR and study chemosensitivity. Minimally toxic concentrations of the ER stress inducer thapsigargin protected cells from cytotoxic agents, with a reduction in antiproliferative drug effect. The activity of the novel small molecule versipelostatin, reported to downregulate the ER molecular chaperones GRP78 and GRP94, was also investigated, with the downregulation previously reported in solid tumour cell lines (Park et al. 2004) confirmed in HT-29 cells, but not observed in the haematological cell lines studied (although versipelostatin was an effective cytotoxic agent at low micromolar concentration). Combination experiments with the chemical chaperone 4-phenylbutyric acid (PBA) resulted in a small increase in apoptosis when PBA was combined with ER stress inducers. However, PBA also showed HDAC inhibitory activity at the concentrations used. Finally, siRNA mediated silencing of GRP78 and GRP94 in THP1 (AML) and U266 (myeloma) cells resulted in a decrease in the targeted protein, but showed only minimal effects on chemosensitivity. In conclusion, the UPR is activated in these haematological cancer cell lines and plays a complex role in chemosensitivity. In contrast to previous reports in solid tumour cells, modulating the UPR in these haematological malignancies had only a modest effect on chemosensitivity.

Multiple Myeloma (MM) is an incurable plasma cell malignancy and, although novel treatment regimes in the past decade have improved patient outcome, long-term treatment leads to relapse and refractory disease. The centrosomal kinase NEK2 is found overexpressed in MM and promotes chromosomal instability, drug resistance and increased proliferation. Although much research shows NEK2 having a detrimental effect in cancer, much of its mechanisms of overexpression and drug resistance has not been studied in detail. In this work we expand our understanding of NEK2 in MM. Using Tandem Affinity Purification coupled with Mass Spectrometry, we show that NEK2 directly interacts with the de-ubiquitinase USP7. We confirm this interaction in cell lines of MM and lung cancer. Since USP7 has been shown to have important cancer-promoting roles we tested if USP7 was necessary for NEK2-driven bortezomib resistance. We found that USP7 shRNA was sufficient to sensitize the bortezomib resistant NEK2 overexpressing cells to bortezomib. Surprisingly, we found that USP7 inhibition with shRNA or by treatment with the small molecule USP7 inhibitor P5091 led to depletion of NEK2 protein in every cell line tested. Previous research shows USP7’s main function is a de-ubiquitinase and, since NEK2 is a target of the ubiquitin-proteasome system, we hypothesized USP7 may be de-ubiquitinating NEK2. Through western blots and immunoprecipitations, we show the NEK2-USP7 interaction promotes the de-ubiquitination and subsequent stabilization of NEK2, presenting USP7 as the first discovered de-ubiquitinating enzyme of NEK2. To understand how NEK2 promotes drug resistance in cancer we studied a previously published list of NEK2-regulated genes and, using the UCSC genome browser (Track Name:GM12878+TNFa RELA) ChIP-seq data, we found approximately half of these genes have the NF-κB transcription factor p65 bound throughout the gene sequence. We also produced a signaling score using an average of 11 known targets of NF-κB and patients with high NEK2 showed a significantly increased score of NF-κB signaling. Additionally, through western blots and immunofluorescence, we found that patients with high NEK2 protein levels consisitently had activation higher signal of p65 protein and phosphorylated p65 at Serine 536, indicative of increased activity. We then causally show NEK2 activates canonical NF-κB by performing western blots and a dual-luciferase reporter assay on control and NEK2 overexpressing cells. Using AKT and PP1α inhibitors, we found that NEK2 drives NF-κB by phosphorylating and inactivating PP1α, leading to hyperactive AKT. Using this model of NEK2-NF-κB activation, we aimed at targeting NEK2 directly with the small molecule drugs INH1 (depletes NEK2 protein) and P5091 (inhibits USP7 activity) in empty vector control cells, NEK2 overexpressing cells or cells with an acquired drug resistance phenotype. Our results show that both INH1 and P5091 can overcome bortezomib resistance in cell lines and in vivo. Another aspect of MM disease we targeted in this work was bone disease. Bone disease in MM is common and causes bone pain and fractures but a much is still regarding what drives these lesions. We found that NEK2 expression in patients correlates with a presence of bone lesions, based on FDG-PET scan and MRI. Using our previously published list of NEK2 regulated genes, we found Heparanase (HPSE) is directly correlated to NEK2 expression. HPSE is an extracellular protein shown to promote differentiation of the bone destroying cell, osteoclast. Using western blots, RT-qPCR and ELISA, we found NEK2 increases HPSE expression and extracellular release. HPSE was also on the list of genes upregulated by NEK2 found to have p65 bound to the gene, thus we tested if NEK2 was driving HPSE through the NF-κB. Accordingly, we found NEK2 drives HPSE through the NF-κB pathway and, consistent with our previous results, in a USP7-dependent manner. Using bone marrow macrophages and conditioned media from empty vector control or NEK2 overexpressing cells, we found NEK2 promtoes increased differentiation of osteoclasts and inhibition of HPSE blocked this effect, strongly suggesting HPSE is the mediator of this effects. Importantly these findings were recapitulated in vivo. Empty vector or NEK2 overexpressing cells were injected through the tail vein to allow dissemination to the bone marrow. microCT and Xray revealed mice injected with NEK2 overexpressing cells showed reduced bone density, compared to empty vector cells. Additionally, H&E and TRAP staining confirmed our in vitro results by showing higher osteoclast levels in bone sections of mice injected with NEK2 overexpressing cells. Lastly, we show a novel role for the ATPase TRIP13 as a cofactor for USP7 de-ubiquitinating activity. TRIP13 is overexpressed in cancer, has been shown to be an oncogene and promotes drug resistance. By systematically targeting TRIP13 overexpressing cells with drugs that inhibit different pathways we found TRIP13 drug resistance is diminished by inhibiting USP7. We found that TRIP13 binds with USP7 and by western blots and immunoprecipitations we show it is necessary for the de-ubiquitination of NEK2. Furthermore, we also found TRIP13 shows a hyperactive USP7 phenotype, shuttling PTEN out of the nucleus and stabilizing MDM2, in a USP7 dependent manner. In summary, this work shows the de-ubiquitinase USP7, coupled with the ATPase TRIP13 stabilizes NEK2 by de-ubiquitination, this leads to accumulation of NEK2 and activation of the canonical NF-κB pathway through PP1α/AKT, which promotes drug resistance and activates HPSE, increasing osteoclast differentiation and bone destruction.

Emerging evidence indicates that 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression in transgenic mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development in genetically engineered mouse models of liquid human cancers - including neoplasms of immunoglobulin (Ig)-producing plasma cells - has not been established. Here we use a double-transgenic strain of laboratory mice, designated C.IL6Myc, that recapitulates key features of human plasma cell myeloma (a.k.a. multiple myeloma [MM]) to demonstrate that FDG-PET/CT affords a useful research tool for assessing plasma cell tumor (PCT) development in a serial, objective and, importantly, stage- and lesion-specific manner. Supported by serum biomarker analyses (Ig level, paraprotein) and histopathological findings in C.IL6Myc mice undergoing PCT development, the newly generated FDG-PET/CT data set demonstrates the potential of this imaging modality for preclinical basic and translational MM research. PET imaging of genetically engineered mice in which MM-like tumors arise predictably in an intact immunocompetent microenvironment may facilitate the design and testing of new approaches to the treatment and prevention of MM in humans.

"August 2004" / Errata inside front cover. / Bibliography: leaves 126-159. / xi, 159, [12] leaves : ill., plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine and The Hanson Centre, Institute of Medical and Veterinary Science, 2004

Multiple myeloma is a malignant bone marrow plasma cell tumor which is responsible for approximately 12,000 deaths per year in the United States and two percent of all cancer deaths. It is recognized clinically by the presence of more than ten percent bone marrow plasma cells, the detection of a monoclonal protein (M-protein), anemia, hypercalcemia, renal insufficiency, and lytic bone lesions. The disease is usually preceded by a premalignant tumor called monoclonal gammopathy of undetermined significance (MGUS), which is present in one percent of adults over the age of fifty, three percent over the age of seventy and ten percent of those in the tenth decade. MGUS is also recognized by the detection of M-protein, but with less than ten percent bone marrow plasma cells and without the other features exhibited by myeloma. The majority of MGUS patients remain stable for long periods without ever developing myeloma. Only a small percentage of patients with MGUS eventually develop multiple myeloma. However, the reason for this is not yet known. Once the myeloma stage is reached, a sequence of well-understood mutational evets eventually lead to the escape of the tumor from the control of the immune system. We propose a mathematical model of tumor-immune system interactions at the onset of the disease in an effort to better understand the early events that take place and their influence on the outcome of the disease. The model is calibrated with parameter values obtained from available data and we study the resulting dynamics. Next, we study how the behavior of the system is affected as parameters are varied. Finally, we interpret the results and draw some conclusions.

Epigenetics continues to be redefined and new discoveries are likely to revolutionise the field still further. This thesis explores different aspects of how epigenetic regulation of gene expression contributes to human disease. Paper I explores the function of the IKKα kinase in regulating gene expression through the nuclear retinoic acid receptor (RAR). We define a set of genes requiring IKKα for their expression and found recruitment of IKKα to the RAR dependent on structural motifs in its protein sequence. This interplay between the NFκB pathway and nuclear receptor regulated transcription is important to consider when designing therapeutic strategies. Papers II and III focus on the plasma cell malignancy multiple myeloma (MM) and define a gene regulatory circuit defining an underexpressed gene profile in MM dependent on the Polycomb proteins. We provide proof-of-principle that the use of small chemical inhibitors may be operational in reactivating genes silenced by H3K27me3 and that this leads to decreased tumour load and increased survival in the 5T33 in vivo model of MM. We explored the genome-wide distribution of H3K27me3 and H3K4me3, and defined their association with gene expression in freshly-isolated malignant plasma cells from MM patients. Importantly, H3K27me3-marked genes in MM associated with more aggressive stages of the disease and less favourable survival. We present evidence that gene targeting by H3K27me3 is likely to not only involve a small population of tumour cells, but rather represent a common MM profile and further provide a rationale for evaluating epigenetic therapeutics in MM. Paper IV shows that pro-inflammatory gene expression in monocytes of psychiatric patients can be induced in vitro by sodium pump inhibitors, as the steroid hormone ouabain. We suggest that the ouabain-induced gene expression is regulated by an intricate network involving microRNAs, Polycomb and the H3K27me3 demethylase JMJD3. Our data indicates that epigenetic regulators play a role in transmitting cues between intrinsic and/extrinsic stimuli and gene expression in psychiatric illness. This thesis provides novel insights on how seemingly unrelated pathways may converge on transcriptional regulation and evidence that epigenetic modifiers contribute to the pathogenesis of human complex diseases such as multiple myeloma and mood disorders.