A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity

Abstract

Multiple myeloma (MM) is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to MM progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIFs), and targeting HIFs can have anti-cancer effects in both solid and hematological malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent and this could be explained by the differential expression of the regulatory prolyl-hydroxylase isoforms. The anti-MM effects of a sequence-specific DNA-binding pyrrole-imidazole polyamide (HIF-PA), that disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable to that of HIF transcription factors. Most of the MM cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O2-dependent activation of HIFα using polyamides.

Restoration of the prolyl-hydroxylase domain protein-3 oxygen-sensing mechanism is responsible for regulation of HIF2α expression and induction of sensitivity of myeloma cells to hypoxia-mediated apoptosis

“…We previously analyzed the expression patterns for HIF1α- and HIF2α-subunits in a panel of MM cell lines and found that HIF1α expression was mostly O 2 -dependent and expression levels were strongly induced by hypoxia, whilst HIF2α tended to be constitutively expressed (under both normoxic and hypoxic conditions) and this tended to be independent of O 2 levels [ 9 ] ( Fig 1A ). This implicated that differential regulation of the α-subunits is due to specific variations in the O 2 -sensing pathways so we assayed for expression of the two most common PHD isoforms, PHD2 and PHD3.…”

“…As shown in Fig 1B , OPM2 cells were markedly more sensitive than 8226 to apoptosis (measured by cleavage of PARP and caspase 9) that was induced by culturing the cells under hypoxic conditions, perhaps because both HIF1α and HIF2α are constitutively expressed in 8226 cells. We tested this by using siRNA to target either HIF1α or HIF2α in 8226 cells (as described in [ 9 ]) and assayed the impact of silencing each subunit on the regulation of sensitivity to hypoxia. We have previously shown that siRNA silenced their targeted HIF gene by approximately 75–90% under both normoxic and hypoxic conditions.…”

“…We previously demonstrated that targeting the mTOR signaling pathway with the mTOR inhibitor, rapamycin, in combination with a HIF/DNA targeting polyamide compound (HIF-PA), synergistically killed 8226 MM cells under both normoxic and hypoxic conditions [ 9 , 25 , 26 ]. We next asked if altering PHD3 expression regulated the sensitivity to rapamycin in our model.…”

“…We hypothesized that disruption of the O 2 -sensing pathway(s) and the constitutive activation of HIF may play a critical role in these events by inducing more malignant tumor phenotypes that facilitate migration and engraftment of tumor cells into the hypoxic marrow microenvironment and impact on MM pathophysiology. Highlighting this, we recently demonstrated that HIFα-subunits are differentially regulated by low pO 2 in MM cell lines and that displacing HIF from its DNA-binding site [ 8 ] using a Pyrrole-Imidazole (Py-Im) polyamide compound (HIF-PA) inhibited a subset of HIF-activated genes and sensitized MM cells to hypoxia-mediated apoptosis in vitro and in vivo [ 9 ]. We also found that using a MM xenograft model, targeting HIF activity with polyamides decreased microvessel density and significantly increased the regions of necrosis and apoptosis within the tumor nodules.…”

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

“…One such class of drugs that have been shown to target nucleotide moieties are polyamides. Several polyamides have been developed as inhibitors of gene expression and other biological effectors that bind double-stranded DNA in a sequence-specific manner (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), and other polyamides have also been developed as broad-spectrum antivirals for papillomaviruses and polyomaviruses precisely because they do not show sequence-specific binding properties (19)(20)(21)(22)(23)(24)(25); J. K. Bashkin et al, unpublished data). Others have also since discovered nonspecific polyamide effects (26,27).…”