The Max b-HLH-LZ Can Transduce into Cells and Inhibit c-Myc Transcriptional Activities

Abstract

The inhibition of the functions of c-Myc (endogenous and oncogenic) was recently shown to provide a spectacular therapeutic index in cancer mouse models, with complete tumor regression and minimal side-effects in normal tissues. This was achieved by the systemic and conditional expression of omomyc, the cDNA of a designed mutant of the b-HLH-LZ of c-Myc named Omomyc. The overall mode of action of Omomyc consists in the sequestration of Max and the concomitant competition of the Omomyc/Max complex with the endogenous c-Myc/Max heterodimer. This leads to the inhibition of the transactivation of Myc target genes involved in proliferation and metabolism. While this body of work has provided extraordinary insights to guide the future development of new cancer therapies that target c-Myc, Omomyc itself is not a therapeutic agent. In this context, we sought to exploit the use of a b-HLH-LZ to inhibit c-Myc in a cancer cell line in a more direct fashion. We demonstrate that the b-HLH-LZ domain of Max (Max*) behaves as a bona fide protein transduction domain (PTD) that can efficiently transduce across cellular membrane via through endocytosis and translocate to the nucleus. In addition, we show that the treatment of HeLa cells with Max* leads to a reduction of metabolism and proliferation rate. Accordingly, we observe a decrease of the population of HeLa cells in S phase, an accumulation in G1/G0 and the induction of apoptosis. In agreement with these phenotypic changes, we show by q-RT-PCR that the treatment of HeLa cells with Max* leads to the activation of the transcription c-Myc repressed genes as well as the repression of the expression of c-Myc activated genes. In addition to the novel discovery that the Max b-HLH-LZ is a PTD, our findings open up new avenues and strategies for the direct inhibition of c-Myc with b-HLH-LZ analogs.

Biophysical characterization of the b-HLH-LZ of ΔMax, an alternatively spliced isoform of Max found in tumor cells: Towards the validation of a tumor suppressor role for the Max homodimers

“…Conversely, the homodimerization of Max and the ability of the homodimer to bind DNA (specific and non-specific) with high affinity limits the access of c-Myc/Max to DNA [9,23,26]. Hence, the overexpression of WT Max in GBM cells can be expected to reduce c-Myc transcriptional activities and proliferation, consistent with results reported by others [12–14,33]. …”

Miz-1 and Max compete to engage c-Myc: implication for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1

“…Indeed, our results suggest that the accumulation of Miz‐1 will lead to the sequestration of c‐Myc through the formation of a specific complex between its second MID and the b‐HLH‐LZ of c‐Myc. This also will lead to a population shift from the c‐Myc/Max heterodimer toward the repressive Max/Max homodimers and potentially Max/Mad heterodimers if the Mad proteins are expressed. These dimers could replace the c‐Myc/Max heterodimers located at TSS and lead to the attenuation of c‐Myc transcriptional amplification.…”

Finding MYCure

“…[5][6][7][8] Despite this undisputable therapeutic opportunity, though, Omomyc was deemed too bulky and unfit to ever become a drug. 9 And this is where our last publication is really proving this assumption wrong. Cell-penetrating peptides (CPPs), also known as protein transduction domains, have the ability to allow intracellular delivery of multiple cargos, 10 and have recently received considerable attention because of their high transduction efficiency and low cytotoxicity.…”