Neoadjuvant administration of hydroxychloroquine in a phase 1 clinical trial induced plasma Par-4 levels and apoptosis in diverse tumors

Peng Wang, Ravshan Burikhanov, Rani Jayswal, Heidi L Weiss, Susanne M Arnold, John L Villano, Vivek M Rangnekar, Peng Wang, Ravshan Burikhanov, Rani Jayswal, Heidi L Weiss, Susanne M Arnold, John L Villano, Vivek M Rangnekar

Abstract

Chloroquine and hydroxychloroquine (HCQ) are robust inducers of the tumor suppressor Par-4 secretion from normal cells. Secreted Par-4 causes paracrine apoptosis of tumor cells and inhibits metastasis in mice. We report the clinical results with pharmacodynamic analyses of our Phase I trial using neoadjuvant administration of HCQ in patients with surgically removable early stage solid tumors. This was a single-institution trial of oral HCQ (200 or 400 mg twice daily) given for 14 days prior to planned surgery. Dose escalation was based on isotonic regression to model safety and biological effect based on plasma Par-4 analysis. Eight of the nine patients treated with HCQ showed elevation in plasma Par-4 levels over basal levels. No toxicities were observed with these dose regimens. The resected tumors from the eight HCQ-treated patients with elevated plasma Par-4 levels, but not the resected tumor from the patient who failed to induce plasma Par-4 levels, exhibited TUNEL-positivity indicative of apoptosis. Resected tumors from all nine HCQ-treated patients showed p62/sequestosome-1 induction indicative of autophagy-inhibition by HCQ. Our findings indicate that both dose levels of HCQ were well-tolerated and that Par-4 secretion but not induction of the autophagy-inhibition marker p62 correlated with apoptosis induction in patients' tumors.

Keywords: Par-4; clinical trial; hydroxychloroquine; tumor apoptosis.

Conflict of interest statement

CONFLICT OF INTEREST VMR has financial interest in Parcure LLC, a start-up company, to develop novel secretagogues of Par-4. The other authors have no financial relationships to disclose.

Figures

Figure 1. Induction of plasma Par-4 in…
Figure 1. Induction of plasma Par-4 in cancer patients treated with HCQ
A. Fold increase of Par-4 levels post-HCQ treatment. Plasma samples from the patients were collected pre-HCQ (Day 0), and Day 14 post HCQ treatment and analyzed by Western blot for Par-4 levels. Fold increase at Day 14 post-treatment relatively to pre-treatment (Day 0) levels is shown. B. Representative western blots for Patient 4, Patient 8, and Patient 9 are shown. Fold increase in Par-4 levels at Day 7 or Day 14 post-HCQ treatment relative to Day 0 pre-treatment levels is indicated.
Figure 2. HCQ induced apoptosis and p62…
Figure 2. HCQ induced apoptosis and p62 levels in tumors
A. Plasma from HCQ-treated patients caused ex vivo apoptosis in cancer cells. Aliquots of pre-treatment (Day 0) or post-HCQ treatment (Day 14) plasma or 10% FBS as control were added to H460 lung cancer cell line. After 24 hours, the cells were scored for apoptosis. Mean of three independent experiments + SD are shown. *P < 0.0001, **P=0.8955. B. HCQ induces apoptosis in patients' tumors. Diagnostic biopsies and paired resected tumor specimen were analyzed by TUNEL assay. TUNEL positive cells were scored relative to total number of cells and percentages are presented. Mean of three separate tumor sections + SD are shown. *P< 0.0001, **P = 0.0012, ***P = 0.0002, by the mixed linear model. For the TUNEL data from patient 8, ****P = 0.0343 by the mixed linear model or P = 0.077 by the paired t-test. C. Representative results of TUNEL assay. Diagnostic biopsy tissues (Pre-treatment) and paired resected tumor specimens (14-day Post-treatment) from the patients were subjected to TUNEL assays. Representative images of tumors from patients 4, 8, and 9 are shown. D. Representative results of p62 IHC staining. Diagnostic biopsy tissue (Pre-treatment) and paired resected tumor specimens (14-day Post-treatment) from the patients were subjected to p62 IHC assays. Representative images of tumors from patients 4, 8, and 9 are shown.

References

    1. Shrestha-Bhattarai T, Rangnekar VM. Cancer-selective apoptotic effects of extracellular and intracellular Par-4. Oncogene. 2010;29:3873–80.
    1. Hebbar N, Wang C, Rangnekar VM. Mechanisms of apoptosis by the tumor suppressor Par-4. J Cell Physiol. 2012;227:3715–21.
    1. El-Guendy N, Zhao Y, Gurumurthy S, Burikhanov R, Rangnekar VM. Identification of a unique core domain of Par-4 sufficient for selective apoptosis induction in cancer cells. Mol Cell Biol. 2003;23:5516–25.
    1. Gurumurthy S, Goswami A, Vasudevan KM, Rangnekar VM. Phosphorylation of Par-4 by protein kinase A is critical for apoptosis. Mol Cell Biol. 2005;25:1146–61.
    1. Alvarez JV, Pan TC, Ruth J, Feng Y, Zhou A, Pant D, Grimley JS, Wandless TJ, Demichele A. I-SPY 1 TRIAL Investigators, and Chodosh LA. Par-4 downregulation promotes breast cancer recurrence by preventing multinucleation following targeted therapy. Cancer Cell. 2013;24:30–44.
    1. Shrestha-Bhattarai T, Hebbar N, Rangnekar VM. Par(−4)oxysm in breast cancer. Cancer Cell. 2013;24:3–5.
    1. Burikhanov R, Zhao Y, Goswami A, Qiu S, Schwarze SR, Rangnekar VM. The tumor suppressor Par-4 activates an extrinsic pathway for apoptosis. Cell. 2009;138:377–88.
    1. Burikhanov R, Hebbar N, Noothi SK, Shukla N, Sledziona J, Araujo N, Kudrimoti M, Wang QJ, Watt DS, Welch DR, Maranchie J, Harada A, Rangnekar VM. Chloroquine-inducible Par-4 secretion is essential for tumor cell apoptosis and inhibition of metastasis. Cell Rep. 2017;18:508–19.
    1. Gump GM, Thorburn A. Autophagy and apoptosis-– what's the connection? Trends Cell Biol. 2011;21:387–92.
    1. Mackenzie AH. Pharmacologic actions of 4-aminoquinoline compounds. Am J Med. 1983;18:5–10.
    1. McChesney EW. Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate. Am J Med. 1983;18:11–8.
    1. Tett SE. Clinical pharmacokinetics of slow-acting antirheumatic drugs. Clin Pharmacokinet. 1993;25:392–407.
    1. Rebecca VW, Amaravadi RK. Emerging strategies to effectively target autophagy in cancer. Oncogene. 2016;35:1–11.
    1. Rosenfeldt MT, O'Prey J, Morton JP, Nixon C, MacKay G, Mrowinska A, Au A, Rai TS, Zheng L, Ridgway R, Adams PD, Anderson KI, Gottlieb E, Sansom OJ, Ryan KM. p53 status determines the role of autophagy in pancreatic tumour development. Nature. 2013;504:296–300.
    1. Boya P, Gonzalez-Polo RA, Poncet D, Andreau K, Vieira HL, Roumier T, Perfettini JL, Kroemer G. Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine. Oncogene. 2003;22:3927–36.
    1. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy. 2012;8:200–12.
    1. Maes H, Kuchnio A, Peric A, Moens S, Nys K, De Bock K, Quaegebeur A, Schoors S, Georgiadou M, Wouters J, Vinckier S, Vankelecom H, Garmyn M, Vion AC, Radtke F, Boulanger C, Gerhardt H, Dejana E, Dewerchin M, Ghesquière B, Annaert W, Agostinis P, Carmeliet P. Tumor vessel normalization by chloroquine independent of autophagy. Cancer Cell. 2014;26:190–206.
    1. Barnard RA, Wittenburg LA, Amaravadi RK, Gustafson DL, Thorburn A, Thamm DH. Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy. 2014;10:1415–25.
    1. Malkin D. Li-Fraumeni syndrome. Genes Cancer. 2011;2:475–84.
    1. Zang Y, Lee JJ, Yuan Y. Adaptive designs for identifying optimal biologic dose for molecularly targeted agents. Clinical Trials. 2014;11:319–27.

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