Medical Oncologist, Gastrointestinal Malignancies

	The University of Texas Health Science Center at San Antonio

Autophagy is a conserved protein degradation process that involves the vacuolar sequestration of long-lived cytoplasmic proteins and organelles into a structure called an autophagosome.

 Autophagosomes fuse with lysosomes, which results in the proteolytic degradation of the cytoplasmic contents.

In eukaryotic cells, autophagy plays an important role in the disposal of damaged organelles and proteins, and serves to generate alternative sources of energy for cell survival during cellular stress through the catabolization of protein substrates.

 The activation of stress response genes such as tumor protein p53 by anticancer therapies can stimulate autophagy in addition to apoptosis.

		Figure 1. Responses to Autophagy-Targeting Therapy

		Treatment with vorinostat and hydroxychloroquine yielded a prolonged partial response in a patient with refractory renal cell carcinoma that has been durable for more than 50 cycles of therapy. MRI scans obtained at baseline and post cycles 10 and 50 (C10 and C50, respectively) are shown.

Although prolonged autophagy can result in cancer cell death, recent investigations suggest that therapy-induced autophagy is a reversible response that promotes cancer cell survival, and thus may diminish the efficacy of some therapeutic agents.

Therefore, autophagy may significantly contribute to resistance to a number of anticancer therapeutic modalities.

Our institution chose to study the role of histone deactelyase (HDAC) inhibitors, which are known to induce autophagy, to investigate the impact of targeting the process.

CTRC researchers showed that HDAC inhibitor— induced autophagy blunts its anticancer activity.6 Yet, genetic or pharmacologic disruption of autophagy synergistically modulated the proapoptotic and cytostatic effects of the HDAC inhibitor vorinostat in models of imatinib-resistant chronic myeloid leukemia and colon cancer.

 A therapy-induced increase in the levels of lysosomal protease cathepsin D was identified as a key downstream pharmacodynamic mediator of the proapoptotic effects of the combination of vorinostat and the autophagy inhibitor, chloroquine.

		Figure 2. Schema for Phase II Autophagy Study

		HCQ indicates hydroxychloroquine; mCRC, metastatic colorectal cancer; po, by mouth; RGF, regorafenib; VOR, vorinostat.

Investigators at our institution have shown that autophagy inhibition with hydroxychloroquine enhanced vorinostat-induced apoptosis via ubiquitinated protein accumulation in preclinical cancer models.

When this was translated to the clinic in a phase I study of 31 patients previously heavily pretreated with chemotherapeutic agents, the most common treatment-related toxicities were primarily grade 1-2 nausea, diarrhea, fatigue, anorexia, weight loss, anemia, and creatinine elevation.

 Most common significant toxicities were fatigue in three patients, with anemia, thrombocytopenia, and neutropenia in one patient each.

The maximum tolerated dose of the combination therapy is hydroxychloroquine 600 mg by mouth daily with vorinostat 400 mg daily. One patient with renal cell carcinoma who had failed seven lines of prior therapy had a confirmed durable partial response (RECIST 1.0 criteria, active on study with 50+ cycles) 

-mutated colorectal cancer (CRC) had prolonged stable disease (>4 cycles).

The addition of hydroxychloroquine did not significantly impact the pharmacokinetic profile of vorinostat.

These promising data have led to the opening of a phase II randomized study comparing autophagy inhibition with vorinostat and hydroxychloroquine with regorafenib, the current standard in patients with CRC who are failing standard therapies 

 This study, which has a primary endpoint of progression-free survival, is currently open to enrollment. Preplanned pharmacodynamic analyses will help us identify which patients with refractory metastatic CRC benefit from autophagy inhibition versus angiogenic inhibition.

		Table. Open Phase II Clinical Trials of Autophagy Modulation in Malignancies

		CQ indicates chloroquine; HCQ, hydroxychloroquine; IL-2, interleukin-2 (aldesleukin).

If the early clinical finding holds true for survival benefit in patients with advanced CRC with minimal toxicity to patients, then autophagy modulation with vorinostat and hydroxychloroquine could one day become a treatment option for patients with advanced CRC. This study could result in better treatment choices for patients with refractory metastatic CRC and lay the foundation for future studies of a new class of therapeutics in metastatic CRC. In addition to CRC, autophagy modulation has demonstrated the potential to improve the treatment efficacy of other chemotherapeutic and targeted agents in other cancers.

 Many clinical trials of autophagy modulators are currently recruiting cancer patients, with several ongoing phase II studies in refractory malignancies 

Preliminary results demonstrate tolerability of autophagy modulators with chemotherapy. Final survival and pharmacodynamic analyses of these studies will allow us to better understand the therapeutic potential of autophagy modulation across a variety of cancer types.

			Louis M, Rosato RR, Brauit L, et al. The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress. 

			Li H, Wu X. Histone deacetylase inhibitor, Trichostatin A, activates p21WAF1/CIP1 expression through downregulation of c-myc and release of the repression of c-myc from the promoter in human cervical cancer cells. 

			Kuefer R, Hofer MD, Altug V, et al. Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer. 

			Strait KA, Dabbas B, Hammond EH, et al. Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins. 

			Duan H, Heckman CA, Boxer LM. Histone deacetylase inhibitors down-regulate bcl-2 expression and induce apoptosis in t(14;18) lymphomas. 

			Carew JS, Nawrocki ST, Cleveland JL. Modulating autophagy for therapeutic benefit. 

			Carew JS, Medina EC, Esquivel II JA, et al. Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. 

			Carew JS, Nawrocki ST, Kahue CN, et al. et al. Targeting autophagy augments the anticancer activity of the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance. 

			Mahalingam D, Mita M, Sarantopoulos J, et al. Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors. 

			NIH Clinical Trails Registray. www.ClinicalTrials.gov. Identifier: NCT02316340.

			Galluzzi L, Pietrocola F, Bravo-San Pedro JM, et al. Autophagy in malignant transformation and cancer progression. 

Articles

Although prolonged autophagy can result in
cancer cell death, recent investigations suggest
that therapy-induced autophagy is a reversible
response that promotes cancer cell survival, and
thus may diminish the efficacy of some therapeutic
agents.

Autophagy Modulation May Be a Novel Approach to Treatment of Advanced Cancers

	The University of Texas Health Science Center

Prodrug chemotherapy is an exciting approach by which higher concentrations of cytotoxic or biologically active agents can be achieved at a tumor site while avoiding the systemic toxicity of a non-cell-specific toxin. One promising example of this strategy is the development of the cytotoxic agent thapsigargin, which is derived from the Thapsia garganica plant once known in Mediterranean cultures as the “death carrot.” Efforts to engineer this agent into an effective therapy for hepatocellular carcinoma (HCC) are advancing.

Thapsigargin can kill a broad spectrum of cancer cells but with detrimental effects on normal endothelial cells, fibroblasts, and osteoblasts. The molecular mechanism of action is attributed to the induction of apoptosis (cell death) by a pronounced increase in cytosolic calcium due to blockade of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump to which thapsigargin binds with high affinity.

Due to the highly toxic characteristics of thapsigargin, the challenge was how to deliver the agent to tumors while causing the least impact on normal cells and tissue. The answer was to develop a relatively nontoxic form of thapsigargin; that is, the prodrug, which can be converted into the active cytotoxic agent at a particular location, specifically at the tumor site, for effective anticancer treatment.

In this pursuit, G-202 was generated by coupling a prostate-specific membrane antigen (PSMA)-specific peptide to an analogue of the potent thapsigargin. The daunting task was to identify a tumor-specific protein from which an appropriate peptide could be linked to the drug and make the drug inactive until the peptide could be cleaved from the drug only at the tumor site by that specific protein, such as PSMA, a glutamate carboxypeptidase type II enzyme that cleaves the acidic amino acids glutamate and aspartate. PSMA was of interest because of its unique presence in prostate cancer and most tumor endothelial cells, but not in normal vasculature or normal tissue epithelium. Of note, PSMA is also found in the vasculature of 90% of HCC tumors.

The immense promise of G-202 as a future practice- changing therapy is apparent because this compound has biologic activity only after proteolytic cleavage by PSMA occurs at the tumor site. Normal healthy cells are no more susceptible to unwanted cytotoxic activity because thapsigargin is blocked by the masking peptide, which is selectively cleaved by PSMA in PSMA-positive human tumors such as prostate and HCC tumors. Within the neovasculature, the nontoxic prodrug G-202 is converted by hydrolysis into the active cytotoxic analogue of thapsigargin, 12ADT-Asp, which has the ability to trigger apoptosis by a mechanism similar to that of thapsigargin. Compared with thapsigargin alone, G-202 can achieve higher concentrations of the active agent at the tumor site while avoiding systemic toxicity.

When tested against a panel of human cancer xenografts in vivo, G-202 produced substantial tumor regression at doses that were minimally toxic to the host. These encouraging results led to a phase I open-label, single-arm, dose-escalation study in patients with advanced cancer. The study was designed to evaluate the safety and pharmacokinetics of G-202 as well as to identify a phase II dosing regimen. GenSpera, Inc, based in San Antonio, is sponsoring the research.

In all, 44 patients were recruited for the study at three US institutions, including the Cancer Therapy & Research Center at UT Health Science Center in San Antonio, which was the lead patient enrollment site. Of the participants, 28 were included in the dose-escalation portion of the study and 16 were part of an expanded cohort. Results were presented at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

Most of the toxicities reported were mild and of grade 1 severity, with some patients experiencing increased creatinine/acute kidney injury/acute renal failure, nausea, and infusion-related reaction, ameliorated by prophylactic IV hydration, IV antiemetics, and steroid administration on the day of infusion. Five patients with HCC were enrolled in the expansion cohort. This group averaged 6 cycles of treatment per patient (range, 2-12 cycles), for a total of 29 cycles among all patients. An encouraging outcome was the observation of stable disease for more than 9 cycles in each of 2 patients.

This has led to the opening of a multicenter phase II study evaluating G-202 as second-line therapy for patients with HCC after treatment failure with sorafenib (Nexavar).

 If the early clinical finding holds true in controlling progression of HCC with minimal toxicity to patients, and a successful clinical development program is completed, G-202 could one day become a treatment option for patients with advanced HCC.

New therapies are urgently needed for this patient population. HCC ranks third in cancer deaths worldwide, with ~360,000 deaths annually. The majority (75%) of the world’s patients with HCC are in Asia-Pacific Rim countries. In the United States, the incidence of HCC is increasing and has risen at a faster rate in the Latino/Hispanic population than among non-Hispanic whites. Currently, the only FDA-approved drug for HCC is sorafenib, which gained an indication for unresectable HCC in 2007 after clinical trial findings demonstrated a 3-month improvement in overall survival versus placebo.

			Mahalingam D, Cetnar J, Wilding G, et al. A first-in-human, phase I clinical study of G-202, a thapsigargin-based prostate- specific membrane antigen (PSMA) activated prodrug, in patients with advanced solid tumors. Poster presented at: AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; October 19-23, 2013; Boston MA. Abstract B244.

			NIH Clinical Trials Registry. www.ClinicalTrials.gov. NCT01777594.

Prodrug chemotherapy is an exciting approach by which higher concentrations of cytotoxic or biologically active agents can be achieved at a tumor site while avoiding the systemic toxicity of a non-cell-specific toxin.

Devalingam Mahalingam, MD, PhD

Articles

Autophagy Modulation May Be a Novel Approach to Treatment of Advanced Cancers

G-202 Represents Novel Approach for Attacking Advanced HCC