Combination of Nab-Rapamycin and Perifosine Induces Synergistic Cytotoxicity and Antitumor Activity Via Autophagy and Apoptosis in Multiple Myeloma (MM)
Authors: Cirstea Diana , Hideshima Teru , Pozzi Samantha , Vallet Sonia , Ikeda Hiroshi , Santo Loredana , Rodig Scott , Vaghela Nileshwari , Currie Treeve , Okawa Yutaka , Chhetri Gaurav , Patel Chirayu G. , Perrone Giulia , Gorgun Gullu , Calabrese Elisabetta , Trieu Voung , Desai Neil , Sportelli Peter , Munshi Nikhil C. , Anderson Kenneth C , Raje Noopur 
Published: October 2019

Abstract
Multiple studies have shown the importance of PI3K/Akt/mTOR pathway in the pathogenesis of multiple myeloma (MM). Specifically, it mediates cell proliferation and development of drug resistance. Identifying mTOR as a key kinase acting downstream of Akt led to the prediction that rapamycin, a universal inhibitor of mTORC1-dependent S6K1 phosphorylation, may be useful in the treatment of MM. Cumulative evidence support the hypothesis that rapamycin-induced cytotoxicity is predominantly triggered as a consequence of autophagy (programmed cell death type II) via excessive cell digestion. Therefore activated Akt can be a key upstream inhibitor of two cell death-inducing events: autophagy via mTOR activation and apoptosis via phosphorylation of BAD and inhibition of the catalytic subunit of caspase-9. Importantly, recent studies have demonstrated the existence of strong positive feedback loops from mTOR/S6K1 to Akt, resulting in Akt activation in some cancer types. We confirmed that suppression of mTOR signaling by rapamycin was associated with upregulation of Akt phosphorylation, which may reduce sensitivity to rapamycin. We therefore hypothesized that inhibition of this positive feedback by a potent Akt inhibitor perifosine could augment rapamycin-induced cytotoxicity in MM cells. As expected, perifosine inhibited rapamycin-induced p-Akt, associated with synergistic cytotoxicity, even in the presence of IL-6, IGF-1 or bone marrow stromal cells. Synergism was confirmed by the Chou-Talalay model, demonstrating that a combination index was < 1 over a range of doses evaluated. We further examined the molecular mechanisms whereby this combination treatment induced synergistic cytotoxicity. We first studied its effect on ultrastructure in MM cells assessed by electron microscopy, demonstrating that rapamycin-induced autophagy was augmented by perifosine in MM.1S cells. We also confirmed autophagy by immunofluorescent staining of LC3, an autophagosome membrane-associated protein. Interestingly, both autophagy and apoptosis were observed when cells were cultured for longer periods with rapamycin and perifosine co-treatment. The apoptotic effect of the combination was further assessed by Annexin/PI flow cytometric analysis and caspase/PARP cleavage. Neither inhibition of autophagy by 3-MA, nor the blockade of apoptosis by Z-VAD-FMK rescued MM cell death from the combination treatment. Finally, we demonstrate that the combination of nanoparticle albumin-bound (nab) -rapamycin and perifosine had significant antitumor activity in an in vivo human MM cell xenograft murine model, associated with prolonged survival. We observed pAkt upregulation induced by rapamycin, which was inhibited by perifosine evidenced by immunohistochemical analyses on tumor tissue from the xenograft model. Importantly, autophagy and apoptosis were also confirmed by LC3 and caspase 3 staining on tumor tissue. Collectively our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine co-treatment induces autophagy and apoptosis, resulting in synergistic cytotoxicity in MM, providing the rationale for use of this combination in future clinical trial in patients with MM.