The Combination of the mTOR Inhibitor Rapamycin and Protein Kinase C Inhibitors Produces Marked Synergistic Anti-Myeloma Effects.
Authors: Shalitin Dror , Share Michael , Campbell Richard A. , Steinberg Jeffrey , Sanchez Eric , Wang Joseph , Leuchter Richard , Li Mingjie , Wang Cathy S. , Chen Haiming , Berenson James R. 
Published: November 2019

Abstract
Mammalian target of rapamycin (mTOR) is a central cell regulator involved in cell survival, growth and proliferation, and is being targeted for cancer therapy. There are two mTOR complexes, the rapamycin-sensitive mTORc1, and the rapamycin-insensitive mTORc2, both of which are downstream of the PI3K/Akt pathway. Protein Kinase C (PKC) refers to a family of serine/threonine kinases that are involved in cell growth, differentiation, apoptosis and migration, and regulated by mTORc2. We evaluated the effects of rapamycin in combination with several PKC inhibitors on the mTOR and PI3K/Akt pathways, two major routes involved in survival of multiple myeloma (MM) cells. First, we examined the expression of several key proteins involved in the regulation of these pathways. PTEN, a phosphatase that blocks AKT activation by inhibiting its upstream regulator PI3K, was highly expressed in U266 and RPMI8226, but found at much lower levels in MM1S. TSC1 and TSC2, proteins regulated by Akt, were also found at much lower levels in MM1S when compared to both U266 and RPMI8226. In contrast to the low levels of PTEN, TSC1 and TSC2, MM1S contained very high levels of PKCζ, a kinase that was undetectable in both U266 and RPMI8226. Given that TSC1 and TSC2 have been shown to negatively regulate mTOR, and PKCζ has been shown to be downstream of mTORc2, we examined the survival and proliferation of MM cells exhibiting normal and over-expression of PTEN following treatment with the mTORc1 inhibitor rapamycin. To modify PTEN expression, MM1S cells were transfected with the over-expression pCEP4-PTEN vector or the empty pCEP4 plasmid. The cells were treated with rapamycin (100nM) for 4 hours and then exposed for 10–30 minutes to FBS. Next, total protein was analyzed by immunoblot for expression patterns and phosphorylation events. The phosphorylation by mTORc1 of S6K was markedly suppressed in cells treated with rapamycin, independent of PTEN expression levels. Additionally, PKCζ phosphorylation was upregulated after treatment with rapamycin, also independent of PTEN expression levels. Based on these results, we hypothesized that blocking mTORc1 leads to a feedback response that increases the activity of mTORc2, resulting in heightened PKCζ phosphorylation levels which may enhance tumor cell growth. Thus, we investigated the effects of blocking mTORc2 through inhibiting its downstream effector PKC as well as mTORc1 with rapamycin on MM cell growth and survival. To determine this, we used several PKC inhibitors in combination with rapamycin. In vitro, MM1S, U266 and RPMI8226 were equally sensitive to single agent rapamycin (IC50 20μM) and the PKC inhibitors rottlerin (IC50 3μM) and Gö 6976 (IC50 1μM). Combinations of rapamycin with either rottlerin or Gö 6976 both significantly increased the ability to inhibit cell proliferation in all three cell lines. As calculated by the Chou-Talalay method, marked synergistic anti-MM effects were observed with both PKC inhibitors. Based on our in vitro results, we are currently evaluating the combination of rapamycin and rottlerin in vivo using our SCID-hu MM models. These promising results provide the potential for further exploration of this new combination approach for the treatment of MM.