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Introduction: Bortezomib (B) is the first in class proteasome inhibitor that received FDA approval for the treatment of MM patients with relapsed or refractory disease. Despite impressive clinical activity all patients develop resistance to B. The underlying mechanism of resistance to B remains undetermined. Mechanisms to overcome B resistance or development of new therapeutic agent(s) with activity in context of B resistance are limited due to unavailability of established preclinical B resistant MM models. To overcome this challenge we developed a B resistant HMCL. Methods: we chronically exposed the OPM-2 HMCL to sub-lethal doses of B. Surviving cells were harvested, re-cultured and dose of B incrementally increased over prolong period of time. The resulting resistant cells were further characterized using array Comparative Genomic Hybridization (aCGH), spectral karyotyping (SKY) and gene expression profiling. Results: After several passages we were able to induce B resistance in this HMCL. Final clone (OPM-2BR) demonstrate resistance to 2 × IC50 dose of B. While SKY and aCGH analysis demonstrated significant differences when compared with parent OPM-2WT (wild type) HMCL, gene expression profiling of the resistant and parental lines demonstrated significant upregulation in the expression of a number of ATP-binding cassette transporters. For example, the breast cancer resistance protein (ABCG2) is upregulated 4-fold in the resistant cell line compared to the parental cell line. This data suggests that drug efflux mediated by drug transporters represents one potential mechanism of resistance to B. Conclusion: Chronic in vitro B exposure results in induced resistance in HMCL-OPM-2BR. Resistant cell line demonstrates cytogenetic variability when compared to the parent cell line. Induction of resistance is stable and provides an important preclinical tool to investigate mechanism(s) of B resistance as well as new drug development for B resistant myeloma patients. Detailed analysis of this cell lines including therapeutic interventions investigating resistance reversal will be presented at the meeting.

Introduction: In a phase II clinical trial we reported the antileukemic effect of L in CLL pts. In this clinical trial 24/34 (70.5%) pts with detectable tumor cells (ALC > 5,000) in peripheral blood demonstrated a decrease in ALC within 8 days of treatment. Although, the exact molecular mechanism for its antitumor activity remains undetermined, L has been reported to down regulates production of various cytokines including VEGF, IL-6 and TNF-alpha. In order to investigate the underlying mechanism(s) responsible for the antileukemic effects of L in CLL, we examined down stream targets of VEGF and IL-6 signaling pathways in CLL cells obtained from pts pre and post 8 day treatment with L. Method: Pts with accessible tumor cells in blood (ALC > 5000) who received L were identified. Tumor cells were obtained prior to (baseline) and 8 days after treatment with L. Whole cell lysate was prepared and expression of down stream targets of VEGF, AKT and Erk determined through immunobloting assay. Results: In cells obtained from pts who had received 8 days of treatment, we observed that L decreased the expression of pAKT and pErk1/2 without changes in corresponding total protein. Conversely L did not have any effect on protein expression of the Bcl-2 or Bcl-xl, though Mcl-1 was decreased compared to pretreatment control. Conclusion: This is the first time that the antileukemic effect of L in CLL may at least be attributed to down regulation of VEGF and IL-6 mediated prosurvival pathways. Our observation that L does not interrupt the Bcl-2 pathway are clinically intriguing and propose the possibility of combining L with Bcl-2 inhibiting agent(s) to augment the antitumor response in CLL.

Introduction: L is a more potent analogue of thalidomide with antitumor activity reported in MDS and multiple myeloma. Clinical anti-leukemic activity of L is reported for the first time by our group in pts with CLL. The underlying mechanism of its antitumor activity remains undetermined. We investigated the effect of L on the tumor microenvironment and studied the modulation of soluble cytokines and immune cells (T and NK cells) in pts receiving L. Patients and Methods: CLL pts enrolled on the clinical study with L were eligible. Pre and post (day 7) samples were obtained for evaluation of changes in serum cytokine and immune cell environment. Malignant cells were also obtained to investigate the in vitro antitumor activity of L prior to initiating treatment on clinical trial. Results: With Anexin V staining for evaluation of apoptosis induction, in vitro testing of pts samples (n=10) showed only a modest increase in apoptosis at 200mg of L - levels clinically not achievable. Yet same pts treated with L on clinical study showed significant antitumor response, suggesting the mechanism to be possibly related to modulation of the tumor microenvironment. In evaluation of the tumor cytokine microenvironment (n= 10) we noted significant L induced increase in IL-10 (n=6), IL-8 (n=8), IP-10 (n=10), IL-8 (n=8), TNF-alpha (n=4) and decrease in PDGF (n=5) and RANTES (n=5). While evaluation of the immune cell repertoire we observed an absolute increase in T-cell as well as NK-cell after treatment with L. Conclusion: Our in vitro evaluation does not suggest a direct apoptotic effect of L on the malignant CLL cells and thus support the hypothesis that the anti-leukemic effect noted in the clinical trial (reported separately) is most likely from in vivo modulation of the tumor microenvironment as is demonstrated from changes in the cytokine milieu and the cellular immune response. Collectively these changes may be responsible for the immune modulating properties of L and the resultant anti-CLL activity in pts.