Dr Melissa Ooi

Paper Abstracts:

Resistance mechanisms in Myeloma

PhD Students-Dr Melissa Ooi (2007-2010), Dr Catriona Hayes(2011-2013) NICB, DCU, Dublin, Ireland and DFCI, Boston, USA

The aims of this project are to examine resistance mechanisms to bortezomib in MM including multidrug resistance pumps, p53 signalling perturbations, to examine the AMPK pathway as an alternative target to overcome MM resistance and finally to examine myeloma resistance to bortezomib treatment using comparative proteomics.
Comparative proteomics to investigate myeloma resistance to bortezomib

Currently, advances in the use of proteomic technologies (specifically the comparative proteomic approach) provide a robust approach to study multiple signaling pathways simultaneously and mechanism of resistance to anticancer therapy. The altered proteins identified by proteomic approach can be further characterized as potential drug targets and the global analysis of the protein alterations can result in valuable information to understand the drug resistance mechanisms. We also investigated bortezomib resistance using proteomics. Using this model and technique, we had isolated several novel proteins including Hsp70 and caspase-3 that were modulated by bortezomib treatment. These two proteins are known in the literature to be affected by bortezomib treatment and this finding validates our technique and model. Verification of novel proteins using ELISA, knockdown studies and in vivo validation are underway.

Multidrug resistance pumps as a mechanism of resistance to bortezomib
The cell membrane is the major determinant of cancer drug penetration to sub cellular targets. Cells have evolved complex chemical defence mechanisms to regulate the entry of foreign substances into and out of the cell. Of the known pump mechanisms, p-glycoprotein (P-gp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. Of all these drug efflux transporters, P-gp is the best studied, and considered to be the most important in contributing to drug resistance. Bortezomib, as described earlier, is a novel treatment for MM. The mechanism of resistance to bortezomib is multifactorial and while little is known about the interaction of bortezomib with P-gp, there are indications that overexpression of this pump may contribute to resistance agent. Rumpold et al.[3] showed that knockdown of P-gp resensitises P-gp expressing cells to proteasome inhibitors. Another strategy to overcome P-gp induced resistance is to prevent P-gp from reaching the cell surface after synthesis in the endoplasmic reticulum. Proteosome inhibitors, lactacystin and MG-132 have been shown to inhibit the maturation of P-gp. Bortezomib may be able to do the same if this is a class effect. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications. In our study, we have shown that overexpression of P-gp attenuates bortezomib activity. A combination of P-gp inhibitor and bortezomib is able to overcome resistance seen to bortezomib as it is a P-gp substrate. Bortezomib is also able to downregulate the expression and function of P-gp. In the clinical setting, the combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable combination. Verification of this finding in vivo using immunohistochemical staining of bortezomib sensitive and resistant patients’ bone marrow biopsies is currently in progress.
p53 signalling perturbations as resistance mechanism in MM

Wild-type p53 maintains cellular integrity by inducing apoptosis or cell cycle arrest of cells with DNA damage. Without an intact p53 pathway, damaged cells continue to proliferate, accumulating more and more genetic lesions that can eventually lead to cancer. The role of p53 in cancer is among one of the most extensively studied and its role as a tumor suppressor has been well established. P53 inactivation by either deletion or mutation seems to be a rare event in MM, and is restricted mostly to the late stages of disease progression. However, as MM patients are surviving longer, p53 inactivation is now emerging as an important factor in drug resistance and shorter survival. The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells. This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy.

The AMPK pathway as an alternative target to overcome MM resistance

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase and serves as an energy sensor in all eukaryotic cells. Several groups have reported that activation of AMPK suppresses mTOR signalling by growth factors and amino acids. The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase and a key regulator of protein translation/synthesis and cell growth. Constitutive activation of PI3K-Akt signalling has been reported in many cancers including glioblastoma, melanoma, prostate cancer and haematological malignancies, including myeloma. One of the most important targets of PI3K is activation of Akt which mediates survival, proliferation and growth of tumour cells. AMPK activation is a feasible therapeutic strategy for these cancers since AMPK inhibits mTOR signalling downstream of Akt, and inhibition of mTOR pathway has been reported to inhibit tumour growth and metastasis in experimental animal models as well as in cultured cells. We investigated the direct effects of 2 AMPK activators, metformin and acadesine (5-Aminoimidazole-4-carboxamide-1-β-Dribofuranoside, AICAR), on a panel of multiple myeloma and solid tumor cell lines. Both AMPK activators exerted on all cell lines tested in vitro a dose-dependent growth-suppressive effect that was preserved or even enhanced in the presence of bone marrow stromal cells or osteoclasts. Mitochondrial membrane depolarization and apoptosis were induced by acadesine, but not metformin. Acadesine-induced apoptosis was attenuated by Bcl-2 and enhanced by activated Akt. Both AMPK activators increased phospho-AMPK and suppressed mTOR signaling. Acadesine treatment decreased tumor burden and prolonged survival in a multiple myeloma xenograft model. Two glycolysis inhibitors, 3-bromopyruvate and 2-deoxyglucose, enhanced the pro-apoptotic activity of acadesine. Our data suggest that AMPK activators exert a direct growth-suppressive effect in various malignancies, involving inhibition of mTOR, that can be potentiated by interactions with the local microenvironment, the Akt pathway and glycolysis inhibitors, supporting their clinical evaluation as anticancer agents, especially for malignancies with active Akt/mTOR pathway.

Indicative References
1: Jakubikova J, Adamia S, Kost-Alimova M, Klippel S, Cervi D, Daley JF,
Cholujova D, Kong SY, Leiba M, Blotta S, Ooi M, Delmore J, Laubach J, Richardson
PG, Sedlak J, Anderson KC, Mitsiades CS. Lenalidomide targets clonogenic side
population in multiple myeloma: pathophysiologic and clinical implications.
Blood. 2011 Feb 14. [Epub ahead of print] PubMed PMID: 21321360.

2: McMillin DW, Delmore J, Negri J, Buon L, Jacobs HM, Laubach J, Jakubikova J,
Ooi M, Hayden P, Schlossman R, Munshi NC, Lengauer C, Richardson PG, Anderson KC,
Mitsiades CS. Molecular and cellular effects of multi-targeted cyclin-dependent
kinase inhibition in myeloma: biological and clinical implications. Br J
Haematol. 2011 Feb;152(4):420-32. doi: 10.1111/j.1365-2141.2010.08427.x. Epub
2011 Jan 11. PubMed PMID: 21223249.

3: Ooi MG, Hayden PJ, Kotoula V, McMillin DW, Charalambous E, Daskalaki E, Raje
NS, Munshi NC, Chauhan D, Hideshima T, Buon L, Clynes M, O’Gorman P, Richardson
PG, Mitsiades CS, Anderson KC, Mitsiades N. Interactions of the Hdm2/p53 and
proteasome pathways may enhance the antitumor activity of bortezomib. Clin Cancer
Res. 2009 Dec 1;15(23):7153-60. Epub 2009 Nov 24. PubMed PMID: 19934289.

4: McMillin DW, Ooi M, Delmore J, Negri J, Hayden P, Mitsiades N, Jakubikova J,
Maira SM, Garcia-Echeverria C, Schlossman R, Munshi NC, Richardson PG, Anderson
KC, Mitsiades CS. Antimyeloma activity of the orally bioavailable dual
phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235.
Cancer Res. 2009 Jul 15;69(14):5835-42. Epub 2009 Jul 7. PubMed PMID: 19584292.