Novel therapy of prostate cancer employing a combination of viral-based immunotherapy and a small molecule BH3 mimetic

ABSTRACT

Cancer-selective viral replication and delivery of a therapeutic immunomodulating, cancer-selective killing cytokine (mda-7/IL-24) by means of a new Cancer Terminator Virus (CTV) combined with a small molecule BH3 mimetic holds promise for treating both primary and metastatic hormone refractory prostate cancer (CaP).

CaP is the leading cause of cancer deaths in men in the US despite advances in chemo-, radio- and hormonal-therapies. Although 5-year survival of loco-regional CaP is 100%, survival dramatically decreases to approximately 28% in metastatic CaP. Consequently, it is imperative to establish newer methods to treat advanced CaP. The distinctive mda-7/IL-24 gene, a member of the IL-10 gene family, that selectively induces cancer-specific apoptosis and toxic autophagy¹ not only in primary transduced tumor cells but also through antitumor ‘bystander’ effects^1-3 in non-transduced adjacent and distant cancer cells makes this gene an ideal candidate for treating advanced and metastatic CaP. To enhance further the therapeutic utility of this gene, we developed a conditionally replication-competent Adenovirus (Ad) comprised of a cancer-selective promoter controlling the Ad replication genes E1A and E1B that concomitantly produces mda-7/IL-24, termed a CTV,^3-5 which provides an ideal gene therapy vector for both primary and metastatic cancers.

Previous studies demonstrate that the CCN1 protein or its promoter is upregulated in cancer and responsible for aggressive and malignant phenotypes.⁶ We showed that CCN1 expression was upregulated in the prostate of male Hi-myc mice with CaP as compared to similar age-matched male mice.⁷ CCN1 expression increases with the age of Hi-myc mice, which recapitulates human CaP development, and expression directly correlates with advanced tumor development with significant elevation of CCN1 expression in 6-month old Hi-myc mice with locally invasive CaP. We also found that a truncated CCN1 promoter, tCCN1-Prom, was upregulated in all CaP cells in comparison to normal immortal RWPE-1 prostate epithelial cells. Moreover, the activity of tCCN1-Prom was more prominent in metastatic CaP cell lines, e.g., PC-3, PC-3ML and ARCaP-M, as compared to less-aggressive CaP cells. Interestingly, metastatic CaP cells showed higher promoter activity of tCCN1 in comparison with the cancer-specific PEG-3 promoter.⁸ Based on these observations, we created a new CTV, Ad.tCCN1-E1A-mda-7 (Ad.tCCN1-CTV-m7), in which the tCCN1-Prom drives Ad replication resulting in mda-7/IL-24 transgene expression.

In our published report,⁷ we documented that Ad.tCCN1-CTV-m7 has potent antitumor activity both in vitro and in vivo in CaP models, including immune-competent transgenic Hi-myc mice. In nude mice containing established CaP in both flanks, intratumoral injection of Ad.tCCN1-CTV-m7 in vivo in the CaP tumor on one flank (representing a primary tumor) reduced its growth as well as inhibiting growth in the non-injected distant tumor, confirming ‘bystander’ antitumor activity. Moreover, this inhibitory effect was greater with Ad.tCCN1-CTV-m7 as compared with Ad.PEG-CTV-m7 (a CTV-m7 with PEG-3-Prom utilized for controlling the replication of virus)⁵ that exceeded activity of a replication incompetent Ad.mda-7 (INGN-241—which has performed well in clinical trials).

Major hurdles to effective systemic Ad gene therapy are viral entrapment in the liver and immune clearance. To overcome these impediments to effective Ad gene therapy, we are pioneering and refining an innovative stealth delivery approach ‘ultrasound-targeted microbubble-destruction (UTMD)’.⁵ In this therapeutic strategy, Ads are conjugated with targeted or decorated microbubbles (D-MBs) and treated with complement prior to injection into the tail vein and finally released at the target site by sonoporation using ultrasound. By this approach, Ad.tCCN1-CTV-m7 was successfully delivered in the prostate region of 5–6 month old Hi-myc mice. Expression of MDA-7/IL-24 was detected in the prostate, which lead to apoptosis in the tumors, resulting in diminished tumor size.⁷

Unfortunately, although demonstrating significant activity, developing a “cure” using a single viral gene therapy is unlikely. The therapeutic efficacy of conditionally replication-competent viruses in both preclinical and clinical trials is enhanced when combined with either radio- or chemo-therapy. Based on prior studies showing that specific BH3 mimetics, such as BI-97C1 (Sabutoclax), can enhance the efficacy of mda-7/IL-24-mediated therapy of CaP,⁹ we have explored the use of a novel BH3 mimetic, BI-97D6,¹⁰ with strong affinity for the Bcl-2 family of proteins especially Mcl-1, to enhance Ad.tCCN1-CTV-m7 therapy of CaP. Mcl-1 is highly expressed in CaP and is often associated with therapy resistance. Our results provided support for the hypothesis that overexpression of Mcl-1 may be responsible for therapy resistance to mda-7/IL-24-induced therapy and the addition of BH3 mimetic BI-97D6 that antagonizes the effect of overexpressed Mcl-1 could reverse the inhibitory effect of this protein. We demonstrated that the BH3 mimetic BI-97D6 with higher affinity for Mcl-1 than BI-97C1 promoted enhanced inhibition of in vitro growth of CaP cells compared to BI-97C1, and the addition of mda-7/IL-24 by Ad.tCCN1-CTV-m7 improved further the efficacy of treatment.⁷ The combination of Ad.tCCN1-CTV-m7 and BI-97D6 enhanced anticancer effect by inducing phosphorylation of p-38 and expression of GRP-94 resulting in ER stress, decreased Mcl-1 expression and induced apoptosis as compared to either agent alone. Interestingly, we have also seen that BI-97D6 increased the stability of otherwise unstable mda-7/IL-24 mRNA at the post-transcriptional level, thus enhancing the production of MDA-7/IL-24 protein and concurrently mda-7/IL-24-mediated apoptosis. Our data also supports the use of Ad.tCCN1-CTV-m7 in complex with D-MBs using the UTMD approach to effectively target the prostate gland in immunocompetent Hi-myc mice. This was confirmed by expression of MDA-7/IL-24 protein followed by cell death in CaP sections from Hi-myc mice culminating in diminished prostate tumor size, which was further enhanced by BI-97D6.⁷

In summary, the combination of Ad.tCCN1-CTV-m7 and BI-97D6 promotes multiple cellular changes enhancing the therapy of primary CaP and metastases (Fig. 1). These include: (i) direct oncolytic effects of Ad.tCCN1-CTV-m7 on primary infected tumor cells, (ii) synergistic antitumor effect of BI-97D6 and mda-7/IL-24 on primary infected (treated cancer cells) and distant tumor cells, (iii) antitumor ‘bystander’ effect of secreted MDA-7/IL-24 on uninfected or distantly located tumor (metastatic) cells, (iv) immune enhancing activity of the cytokine MDA-7/IL-24, and (v) inhibitory effect on tumor vasculature of MDA-7/IL-24 by hindering angiogenesis. Considering the safety profile of BH3 mimetics, conditionally replication-competent Ads and successful Phase I clinical trials of mda-7/IL-24, our studies support potential therapeutic utility of a combinatorial approach for the therapy of primary and advanced CaP.

Figure 1.

Combinatorial effect of a Cancer Terminator Virus (Ad.tCCN1-CTV-m7) and a BH3 mimetic (BI-97D6) in eradicating prostate cancer (CaP): Ad.tCCN1-CTV-m7 was mixed with decorated (targeted) microbubbles (MB) specifically targeting and binding to the prostate tumor vasculature that overexpresses VCAM-1, and injected intravenously (by tail vein injection) followed by delivery to the prostate region by the ultrasound-targeted microbubble-destruction (UTMD) approach. Following release and subsequent infection of the CaP cells, Ad.tCCN1-CTV-m7 selectively replicates in CaP cells resulting in production of mda-7/IL-24, which upon translation into protein can be secreted into the circulation or directly kill infected cancer cells. MDA-7/IL-24 can promote mitochondrial intrinsic apoptosis via the Bcl-2-family pathway (as well as extrinsic apoptosis via death receptors), as well as ER stress or toxic autophagy in prostate tumors (primary site of infection). BI-97D6, which displays some antitumor activity in CaP as a single agent, synergistically co-operates with Ad.tCCN1-CTV-m7 in inducing cancer-specific apoptosis. BI-97D6 also stabilized mda-7/IL-24 mRNA further enhancing the production of MDA-7/IL-24 protein. The secreted MDA-7/IL-24 protein acts as a cytokine on adjacent uninfected CaP cells or tumors located distantly (lung, bone metastasis) via receptor dimerization and signal transduction culminating in cancer-specific apoptosis. It can also activate anti-angiogenesis effects and antitumor-immune responses further amplifying the antitumor effects of MDA-7/IL-24, thereby culminating in eradicating the primary CaP as well as any metastasis to distant sites in the body.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.