Purpose We determined the activity of heat shock protein (hsp) 90 inhibitor (HI), and/or JAK2 tyrosine kinase inhibitor (TKI) against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE1) or primary human CD34+ myeloproliferative neoplasm (MPN) cells. Experimental Design Following exposure to the HI AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined. Results Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, i.e., phospho (p)-STAT5, p-AKT and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1 and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone, and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Co-treatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34+ MPN versus normal hematopoietic progenitor cells. As compared to the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE1/TGR cells exhibited significantly higher IC50 values for JAK2-TKI (p <0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the HIs AUY922 and 17-AAG; accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT and Bcl-xL, with concomitant induction of BIM. Conclusions Findings presented here demonstrate that co-treatment with HI and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. Additionally, treatment with HI may overcome resistance to JAK2-TKI in human MPN cells.

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis, while promoting autophagy, which promotes cancer cell survival when apoptosis is compromised. Here, we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex, resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62, caused synergistic cell death of MB-231 and SUM159PT cells, and inhibited mammosphere formation in MB-231 cells, compared with treatment with each agent alone. Finally, in mouse mammary fat pad xenografts of MB-231 cells, a tumor size-dependent induction of heat shock response, ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively, our findings show that cotreatment with an autophagy inhibitor and pan-HDI, for example, chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins, exerts superior inhibitory effects on TNBC cell growth, and increases the survival of TNBC xenografts. Mol Cancer Ther; 11(4); 973-83. Ó2012 AACR.

Hemopoietic progenitor cells

A deregulated epigenome contributes to the transformed phenotype of Mantle Cell Lymphoma (MCL). This involves activity of the PRC (polycomb repressive complex) 2, containing three core proteins EZH2, SUZ12 and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation (3Me) of lysine (K)-27 on histone H3 (3MeK27H3), regulates the expression of HOX genes and promotes cell proliferation and aggressiveness of the transformed cells. Here, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) depletes EZH2, SUZ12 and 3MeK27H3 in the cultured human MCL cells. Treatment with DZNep increased the expression of p21, p27 and FBXO32, while depleting Cyclin D1 and Cyclin E1 levels in MCL cells. Additionally, DZNep treatment induced cell cycle arrest and apoptosis in cultured and primary MCL cells. Further, as compared to treatment with each agent alone, co-treatment with DZNep and the pan-histone deacetylase inhibitor panobinostat (PS) caused greater depletion of EZH2, SUZ12, 3MeK27H3 and Cyclin D1 levels, while inducing greater expression of FBXO32, p16, p21 and p27. Combined treatment with DZNep and PS also synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34+ cells. Co-treatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice. These preclinical in vitro and in vivo findings demonstrate that the co-treatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL.