Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma

Abstract

Histone deacetylases (HDAC) control gene expression through their ability to acetylate proteins, thereby influencing a diverse range of cellular functions. Class I HDAC (HDAC1-3 and 8) and HDAC6 are predominantly upregulated in malignancies and their altered expression in some cancers has a significant prognostic implication. The expression and prognostic consequence of dysregulated Class I HDAC and HDAC6, key players in multiple myeloma (MM), are unknown. This study hypothesized that HDAC are dysregulated in MM and patients with high expression have significantly poorer prognostic outcomes. Quantitative PCR for 11 HDAC (Class I, II, and IV) was performed in genetically heterogeneous human myeloma cell lines (HMCL) and primary MM and compared to normal plasma cells (PC). In HMCL, HDAC1-3 and 8 (Class I), and HDAC5 and HDAC10 (Class II) were significantly upregulated compared to normal PC. In primary MM, the median expression level of all of the HDAC, except HDAC1 and HDAC11, were elevated when compared to normal PC. Patients with higher levels of HDAC1-3, HDAC4, HDAC6, and HDAC11 transcripts demonstrated a significantly shorter progression-free survival (PFS). Immunohistochemical staining for HDAC1 and HDAC6 on bone marrow trephines from a uniformly treated cohort of transplant eligible MM patients revealed that HDAC1 protein was detectable in most patients and that higher levels of MM cell HDAC1 protein expression (≥90 % versus ≤20 % MM cell positivity) correlated with both shorter PFS (P = 0 .07) and shorter overall survival (P = 0 .003). Conversely, while the majority of patients expressed HDAC6, there was no correlation between HDAC6 levels and patient outcome. Together, these results indicate that overexpression of Class I HDAC, particularly HDAC1, is associated with poor prognosis in MM.

Keywords: Class I HDAC; HDAC expression; hematological malignancy; histone deacetylases; multiple myeloma; prognosis; .

Figure 1.

HDAC genes are overexpressed in human myeloma cell lines when compared to normal plasma cells Expression levels of HDAC in 14 human myeloma cell lines (HMCL) were compared to normal plasma cells (normal; n = 9 ). Class I HDAC (HDAC1, HDAC2, HDAC3, and HDAC8) were all significantly overexpressed. Among the class II HDAC, HDAC5 and HDAC10 were significantly overexpressed. Data are presented as mean±SEM. Significant differences were calculated with t-test utilizing GraphPad Prism 5.0d. p-values are indicated.

Figure 2.

HDAC levels are dysregulated in primary MM Expression levels of HDAC in primary MM (n = 55 ) were compared to normal plasma cells (normal; n = 9 ). All genes, except HDAC1 and HDAC11, were significantly altered. Data are presented as median with range. Significant differences were calculated utilizing Mann-Whitney test in GraphPad Prism 5.0d. P-values are indicated.

Figure 3.

Increased HDAC levels correlate to poor prognosis Kaplan-Meir plots showing progression-free survival (PFS) in MM patients with high (>75th centile of MM patient cohort) compared to low (<75th centile) expression profiles. (A-E) Patients with high HDAC1, HDAC2, HDAC4, HDAC6, and HDAC11 expression depicted significantly shorter PFS days than patients with low levels of these genes. Significant differences and median survival days were calculated utilizing GraphPad Prism 5.0d. P-values are indicated.

Figure 4.

Higher HDAC1 levels correlate with poor prognosis (A) Image depicting low staining of HDAC1 (20%) in MM cells; (B) Image depicting high proportion of HDAC1 staining (90%) in MM cells; (C) Kaplan-Meir survival plots for patients with 0–20%, 30–80%, and 90–100% proportion of HDAC1 staining, indicating patients with higher proportion of HDAC1 have shorter progression-free survival days; (D) Kaplan-Meir survival plots for patients with 0–20%, 30–80% and 90–100% proportion of HDAC1 staining indicating patients with higher proportion of HDAC1 have shorter overall survival days. Patients with >70% infiltration of MM cells was chosen for image capture. Significant differences and median survival days were calculated utilizing GraphPad Prism 5.0d and p-values are indicated.

Figure 5.

The majority of patients show high HDAC6 staining in MM cells (A) Image depicting low proportion (30%) and weak staining intensity (+) of HDAC6 in MM cells; (B) Image depicting high proportion (90%) and strong staining intensity of HDAC6 in MM cells; (C) Graph representing the distribution of HDAC6 staining proportion and intensity in bone marrow trephines tested. Higher proportion of HDAC6 staining correlated to stronger staining intensity.