The Hedgehog pathway as targetable vulnerability with 5-azacytidine in myelodysplastic syndrome and acute myeloid leukemia.

BACKGROUND: Therapy and outcome for elderly acute myeloid leukemia (AML) patients has not improved for many years. Similarly, there remains a clinical need to improve response rates in advanced myelodysplastic syndrome (MDS) patients treated with hypomethylating agents, and few combination regimens have shown clinical benefit. We conducted a 5-azacytidine (5-Aza) RNA-interference (RNAi) sensitizer screen to identify gene targets within the commonly deleted regions (CDRs) of chromosomes 5 and 7, whose silencing enhances the activity of 5-Aza. METHODS AND RESULTS: An RNAi silencing screen of 270 genes from the CDRs of chromosomes 5 and 7 was performed in combination with 5-Aza treatment in four AML cell lines (TF-1, THP-1, MDS-L, and HEL). Several genes within the hedgehog pathway (HhP), specifically SHH, SMO, and GLI3, were identified as 5-Aza sensitizing hits. The smoothened (SMO) inhibitors LDE225 (erismodegib) and GDC0449 (vismodegib) showed moderate single-agent activity in AML cell lines. Further studies with erismodegib in combination with 5-Aza demonstrated synergistic activity with combination index (CI) values of 0.48 to 0.71 in seven AML lines. Clonogenic growth of primary patient samples was inhibited to a greater extent in the combination than with single-agent erismodegib or 5-Aza in 55 % (6 of 11) primary patient samples examined. There was no association of the 5-Aza/erismodegib sensitization potential to clinical-cytogenetic features or common myeloid mutations. Activation of the HhP, as determined by greater expression of HhP-related genes, showed less responsiveness to single-agent SMO inhibition, while synergy between both agents was similar regardless of HhP gene expression. In vitro experiments suggested that concurrent dosing showed stronger synergy than sequential dosing. CONCLUSIONS: Inhibition of the HhP with SMO inhibitors in combination with the hypomethylating agent 5-Aza demonstrates synergy in vitro and inhibits long-term repopulation capacity ex vivo in AML and MDS. A clinical trial combining 5-Aza with LDE225 (erismodegib) in MDS and AML is ongoing based on these results as well as additional publications suggesting a role for HhP signaling in myeloid disease.

Tamoxifen prevents apoptosis and follicle loss from cyclophosphamide in cultured rat ovaries.

Recent studies documented that the selective estrogen receptor modulator tamoxifen prevents follicle loss and promotes fertility following in vivo exposure of rodents to irradiation or ovotoxic cancer drugs, cyclophosphamide and doxorubicin. In an effort to characterize the ovarian-sparing mechanisms of tamoxifen in preantral follicle classes, cultured neonatal rat ovaries (Day 4, Sprague Dawley) were treated for 1-7 days with active metabolites of cyclophosphamide (i.e., 4-hydroxycyclophosphamide; CTX) (0, 1, and 10 µM) and tamoxifen (i.e., 4-hydroxytamoxifen; TAM) (0 and 10 µM) in vitro, and both apoptosis and follicle numbers were measured. CTX caused marked follicular apoptosis and follicular loss. TAM treatment decreased follicular loss and apoptosis from CTX in vitro. TAM alone had no effect on these parameters. IGF-1 and IGF-1 receptor were assessed in ovarian tissue showing no impact of TAM or CTX on these endpoints. Targeted mRNA analysis during follicular rescue by TAM revealed decreased expression of multiple genes related to inflammation, including mediators of lipoxygenase and prostaglandin production and signaling (Alox5, Pla2g1b, Ptgfr), cytokine binding (Il1r1, Il2rg ), apoptosis (Tnfrsf1a), second messenger signaling (Mapk1, Mapk14, Plcg1), as well as tissue remodeling and vasodilation (Bdkrb2, Klk15). The results suggest that TAM protects the ovary from CTX-mediated toxicity through direct ovarian actions that oppose follicular loss.