Targeting p53 Via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma

Abstract 1836

The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. We have previously reported the anti-myeloma activity of a small molecule RITA. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. There remain controversies over the notion that RITA increases p53 activity by binding with p53. It is highly possible that that RITA-induced activation of the p53 pathway can also occur in the mechanisms independent of inhibition of the p53-MDM2 interaction.

To expore the molecular signaling pathway in RITA-induced apoptosis, we performe μd gμene expression profiling (GEP), by microarray in MM.1S cells treated with RITA or DMSO control. A significant number of genes (∼46) were associated with different types of stress signaling including p53 and c-Jun N-terminal kinase (JNK) signaling. Consistent with GEP data we found that treatment of H929 and MM.1S cells (harboring wild type p53) with RITA resulted in a dose- and time-dependent increase in the phosphorylation of c-Jun (c-Jun-p) along with up-regulation of p53. Furthermore, we examined the binding of c-Jun to the activator protein (AP-1) binding site of the p53 promoter region. Chromatin immunoprecipitation analysis (ChIP) analysis demonstrated that phosphorylated c-Jun antibody immunoprecipitated a significantly increased proportion of the region of the p53 promoter containing AP-1 site in both MM.1S and H929 cells treated with RITA, whereas the control antibody (IgG) failed to precipitate it. Quantitative analysis by PCR showed a ∼5 and 7-fold increase of c-Jun binding to p53 promoter in RITA-treated MM.1S and H929 cells, respectively, in comparison to DMSO-treated cells.

In order to clarify the involvement of JNK, we investigated the role of JNK in the regulation of p53-mediated apoptosis induced by RITA in MM cells by using a JNK specific inhibitor, SP-600125. SP600125 abrogated the ability of RITA to up-regulate phosphorylated c-Jun and p53. To further understand specific inhibition of JNK activation, using siRNA approach JNK was selectively knocked down in H929 cells resulting in bloackge of RITA-induced activation of c-Jun and p53. Functionally, apoptosis induction by RITA in H929 cells was inhibited by both SP-600125 and JNK siRNA as evidenced by reduction of cleavage of caspase-3 and PARP and attenuation of the RITA-induced increase of Annexin V-positive cells. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also abrogated the activation of c-Jun suggesting the establishment of a positive feedback loop between p53 and JNK. In addition, silencing p53 expression significantly blocked the apoptosis induction by RITA. These results confirm that RITA-induced apoptosis in MM cells is p53-dependent.

Furthermore, we examined the combined cytotoxic effect of RITA and dexamethasone (DXM) or CDDO (both of which are known as JNK activators) in H929 and MM.1S cell lines and primary MM samples. Treatment of H929 cells with RITA or DXM alone induced only 10 to 40% cell killing which was synergistically enhanced to 65% (CI, 0.86) and 80% (CI, 0.55), respectively in RITA plus DXM combination. The combination of 5 μM RITA and 1 μM DXM induced a synergistic cytotoxicity (CI=0.81-0.90) in 3 primary MM samples. In MM.1S cells, the combination of 0.5 μM CDDO with either 0.25 or 0.5 μM RITA displayed a synergistic cytotoxic response with a CI value of 0.83 and 0.62, respectively (p<0.05).

Finally, RITA demonstrates significant anti-tumor activity in a human plasmacytoma xenograft mouse model. Daily intraperitoneal (i.p.) treatment of RITA (10 mg/kg) decreased tumor growth (∼72% inhibition, p<0.01; at day 12 after treatment; n=4) with no apparent toxicity in mice implanted with H929 cells (5 × 10⁶ cells injected s.c. in SCID mice).

These findings reveal a novel mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway. Our study also provides the rationale for combination of p53 activating drugs with JNK activators which may offer improved therapeutic strategies in treating MM patients.