Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.     

SIRT1 is upregulated in cutaneous T-cell lymphoma,and its inhibition induces growth arrest and apoptosis

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs), but has not yet been explored in cutaneous T-cell lymphoma (CTCL). We analyzed five CTCL cell lines and lesional tissues using flow cytometry, immunostaining, immunoblotting, cell death, viability, and apoptosis assays, small-molecule inhibitors, and shRNA knockdown. We found strong SIRT1 expression among CTCL lines relative to normal lymphocytes. CTCL cells in lesional tissues also expressed SIRT1 strongly. SIRT1 knockdown resulted in reduced cellular metabolism and proliferation, increased apoptosis, and PARP cleavage products. Tenovin-1, which reversibly inhibits class III HDACs (SIRT1 and SIRT2), reduced SIRT enzymatic activity and SIRT1 expression and led to increased apoptosis. These alterations were accompanied by increased forkhead box O3 (FoxO3) in several cell lines and increased nuclear p53, as well as acetylated p53 in wtp53 MyLa CTCL line. A combination of class I/II and class III HDACIs (vorinostat and tenovin-1) produced significantly greater growth inhibition, cell death via apoptosis, as well as superior p53 promoter upregulation in wtp53 MyLa cells as compared with either agent alone. This occurred in a partially p53-dependent manner, as these effects were blunted by p53 knockdown. Our results indicate that SIRT1 is strongly expressed in CTCL. Its inhibition results in reduced growth and increased apoptosis of CTCL cells. Furthermore, our findings suggest that some CTCL patients, such as those with wtp53, might benefit more from treatment with a combination of different classes of HDACIs than with a single agent.     

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4⁺ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC₅₀ of 17.38 versus 22.01 μM for cucurbitacin E and 13.36 versus 24.47 μM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC₅₀ of 9.98 and 29.15 μM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.     

Loss of β-arrestin-2 gene and possible functional consequences on Sezary Syndrome

Sezary Syndrome is an aggressive T-cell Lymphoma involving blood, skin and lymphonodes Involvement of the CXCR4-SDF1 has been previously shown. We here present evidence also of the involvement of B-arrestin a downstream regulator of CXCR4, that is depleted and downregulated as well as a potential functional role for this depletion.