Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34⁺ cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34⁺ cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones.

A leukemic in vitro model produced by transducing Cord Blood derived-hematopoietic CD34+ cells with the MLL-AF9 translocation resulting in the oncogenic fusion protein, is used to assess for sensitivity to Zoledronic acid. These cells are practically immortalized and are of myeloid origin. Proliferation, clonogenic and stromal co-culture assays showed that the MLL-AF9 cells were considerably more sensitive to Zoledronic acid than normal hematopoietic CD34+ cells or MS-5 stromal cells. The MLL-AF9 cells were notably more inhibited by Zoledronic acid when cultured as colonies in 3 dimensions, requiring cell-cell contacts compared to suspension expansion cultures. This is coherent with the mechanism of action of Zoledronic acid inhibiting farnesyl diphosphate synthase which results in a block in prenylation of GTPases such that their role in the membrane is compromised for cell-cell contacts. Zoledronic acid can be proposed to target the MLL-AF9 leukemic stem cells before they emerge from the hematopoietic niche, which being in proximity to bone osteoclasts where Zoledronic acid is sequestered can be predicted to result in sufficient levels to result in an anti-leukemic action.

We herein describe the structure-based design, synthesis and biological evaluation of a new class of histone deacetylase 6 (HDAC6) inhibitors guided by X-ray crystal structures of our previously identified compounds 13-15. X-ray co-crystal structures of second-generation inhibitors 19b (Subseries I) and 20b (Subseries II) within zHDAC6 CD2 confirmed that these compounds behave as highly potent HDAC6 inhibitors with the ability to engage the S531 residue in the L2 loop HDAC6 catalytic domain. Western-blot analysis confirmed the target engagement in cell-based settings, through the increase of acetylated tubulin levels, without significant variation in the histone acetylation status. Compound 20b extensively reduced the viability of A549 cells in both 2- and 3-dimensional cell cultures, through the activation of pro-autophagic pathways. In addition, HDAC6 inhibition impaired the nucleation of stress-granules, confirming the ability of these compounds to modulate liquid−liquid phase separation, an emerging HDAC6-driven hallmark in cancer.