Martin Montagne

The Max homodimeric b-HLH-LZ significantly interferes with the specific heterodimerization between the c-Myc and Max b-HLH-LZ in absence of DNA: a quantitative analysis

Specific heterodimerization plays a crucial role in the regulation of the biology of the cell. For example, the specific heterodimerization between the b‐HLH‐LZ transcription factors c‐Myc and Max is a prerequisite for c‐Myc transcriptional activity that leads to cell growth, proliferation and tumorigenesis. On the other hand, the Mad proteins can compete with c‐Myc for Max. The Mad/Max heterodimer antagonizes the effect of the c‐Myc/Max heterodimer. In this contribution, we have focused on the specific heterodimerization between the b‐HLH‐LZ domains of c‐Myc and Max using CD and NMR. While the c‐Myc and Max b‐HLH‐LZ domains are found to preferentially form a heterodimer; we demonstrate for the first time that a significant population of the Max homodimeric b‐HLH‐LZ can also form and hence interferes significantly with the specific heterodimerization. This indicates that the Max/Max homodimer can also interfere with c‐Myc/Max functions, therefore adding to the complexity of the regulation of transcription by the Myc/Max/Mad network. The demonstration of the existence of the homodimeric population was made possible by the application of numerical routines that enable the simulation of composite spectroscopic signal (e.g. CD) as a function of temperature and total concentration of proteins. From a systems biology perspective, our routines may be of general interest as they offer the opportunity to treat many competing equilibriums in order to predict the probability of existence of protein complexes. Copyright

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

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.

Structural Insights into c-Myc Interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding

c-Myc-interacting zinc finger protein-1 (Miz-1) is a poly-Cys2His2 zinc finger (ZF) transcriptional regulator of many cell cycle genes. A Miz-1 DNA sequence consensus has recently been identified and has also unveiled Miz-1 functions in other cellular processes, underscoring its importance in the cell. Miz-1 contains 13 ZFs, but it is unknown why Miz-1 has so many ZFs and whether they recognize and bind DNA sequences in a typical fashion. Here, we used NMR to deduce the role of Miz-1 ZFs 1–4 in detecting the Miz-1 consensus sequence and preventing nonspecific DNA binding. In the construct containing the first 4 ZFs, we observed that ZFs 3 and 4 form an unusual compact and stable structure that restricts their motions. Disruption of this compact structure by an electrostatically mismatched A86K mutation profoundly affected the DNA binding properties of the WT construct. On the one hand, Miz1–4WT was found to bind the Miz-1 DNA consensus sequence weakly and through ZFs 1–3 only. On the other hand, the four ZFs in the structurally destabilized Miz1–4A86K mutant bound to the DNA consensus with a 30-fold increase in affinity (100 nm). The formation of such a thermodynamically stable but nonspecific complex is expected to slow down the rate of DNA scanning by Miz-1 during the search for its consensus sequence. Interestingly, we found that the motif stabilizing the compact structure between ZFs 3 and 4 is conserved and enriched in other long poly-ZF proteins. As discussed in detail, our findings support a general role of compact inter-ZF structures in minimizing the formation of off-target DNA complexes.

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

c‐Myc is a basic helix‐loop‐helix leucine zipper (b‐HLH‐LZ) transcription factor deregulated in the majority of human cancers. As a heterodimer with Max, another b‐HLH‐LZ transcription factor, deregulated and persistent c‐Myc accumulates at transcriptionally active promoters and enhancers and amplifies transcription. This leads to the so‐called transcriptional addiction of tumor cells. Recent studies have showed that c‐Myc transcriptional activities can be reversed by its association with Miz‐1, a POZ transcription factor containing 13 classical zinc fingers. Although evidences have led to suggest that c‐Myc interacts with both Miz‐1 and Max to form a ternary repressive complex, earlier evidences also suggest that Miz‐1 and Max may compete to engage c‐Myc. In such a scenario, the Miz‐1/c‐Myc complex would be the entity responsible for the inhibition of c‐Myc transcriptional amplification. Considering the implications of the Miz‐1/c‐Myc interaction, it is highly important to solve this duality. While two potential c‐Myc interacting domains (hereafter termed MID) have been identified in Miz‐1 by yeast two‐hybrid, with the b‐HLH‐LZ as a bait, the biophysical characterization of these interactions has not been reported so far. Here, we report that the MID located between the 12th and 13th zinc finger of Miz‐1 and the b‐HLH‐LZ of Max compete to form a complex with the b‐HLH‐LZ of c‐Myc. Our results support the notion that the repressive action of Miz‐1 on c‐Myc does not rely on the formation of a ternary complex. The implications of these observations for the mechanism of inhibition of c‐Myc transcriptional activity by Miz‐1 are discussed. Proteins 2017; 85:199–206.

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.

It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max.

Abstract 2167: Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy

Deregulation of the MYC oncoprotein promotes tumorigenesis in most, if not all, cancers and is often associated with poor prognosis. However, targeting MYC has long been considered impossible based on the assumption that it would cause catastrophic side effects in normal tissues. Despite this general preconceived notion, we showed that MYC inhibition exerts extraordinary therapeutic impact in various genetic mouse models of cancer, and causes only mild, well-tolerated and reversible side effects. For these studies we employed the systemic and conditional expression of a dominant negative of MYC, called Omomyc, which we designed and validated, and that can inhibit MYC transactivation function both in vitro and in vivo. To date, Omomyc has only been considered a proof of principle, with any potential clinical application limited to gene therapy. Here we actually show that the 11 kDa Omomyc polypeptide spontaneously transduces into cancer cells, demonstrating unexpected cell-penetrating ability. Once inside the nuclei, the polypeptide effectively blocks MYC binding to its target DNA sites, interfering with MYC transcriptional regulation and halting cell proliferation. Moreover, intranasal (i.n.) administration of the Omomyc polypeptide in mice results in its rapid and persistent distribution to lungs, as well as to other organs (i.e. intestine, liver, kidneys and brain). Importantly, i.n. treatment of mice bearing either Non-Small-Cell-Lung-Cancer (NSCLC) or glioblastoma (GBM) with the Omomyc cell-penetrating peptide (OmomycCPP) significantly reduces tumor burden compared to their control counterparts. Notably, tumor regression is accompanied by significant reprogramming of the tumor microenvironment and tumor immune response. In summary, our data indicate that this novel generation of polypeptides represents a new opportunity to potentially inhibit MYC pharmacologically in a variety of malignant diseases. Citation Format: Marie-eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Peter Rahl, Sandra Martinez-Martin, Loika Maltais, Mariano F. Zacarias-Fluck, Silvia Casacuberta, Erika Serrano del Pozo, Christopher Fiore, Laia Foradada, Matthew Guenther, Eduardo Romero Sanz, Marta Oteo Vives, Cynthia Tremblay, Martin Montagne, Miguel Angel Morcillo Alonso, Jonathan R. Whitfield, Pierre Lavigne, Laura Soucek. Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2167. doi:10.1158/1538-7445.AM2017-2167

Abstract A11: Biophysical characterization of the b-HLH-LZ of deltaMax

Myc proteins (N-, L- and c-Myc) are transcriptional regulators with a broad spectrum of target genes. While essential for development, cell growth and proliferation as well as apoptosis, they play a major role in cancer onset and progression when deregulated. Myc protein functions depend on their heterodimerization with Max, another transcriptional regulator capable of forming a homodimer and antagonizing c-Myc transcriptional regulation. Recent studies have shown that c-Myc can control the alternative splicing of Max9s pre-messenger RNA to produce a c-terminal truncated isoform called deltaMax. The ratio of deltaMax/WT-Max (p21) in cancer cells correlates with increased malignancy. In addition to producing a shorter gene product, the alternative splicing (exon-inclusion) generates a change in sequence in the LZ. The LZ domain is an integral and crucial part of the b-HLH-LZ motif of Max controlling its homodimerization and specific heterodimerization with c-Myc as well as binding to the E-box sequences found in gene promoters and enhancers. Whereas it has been demonstrated that deltaMax increases c-Myc transcriptional activities, the underlying mechanisms have not been elucidated yet. Here, we provide original insights into such mechanisms. First, we show that contrary to the WT motif, the b-HLH-LZ of deltaMax does not homodimerize. In addition, while the b-HLH-LZ of deltaMax can still heterodimerize with the b-HLH-LZ of c-Myc, it can no longer compete with the binding of the heterodimer to E-box sequences. Taken together, our results demonstrate that deltaMax can maximize the amount of c-Myc bound target gene promoters and enhancers and explain how deltaMax and the ratio of deltaMax/WT-Max (p21) in cancer cells can increase c-Myc oncogenic activities. Citation Format: Loika Maltais, Cynthia Tremblay, Mikael Bedard, Martin Montagne, Pierre Lavigne. Biophysical characterization of the b-HLH-LZ of deltaMax. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A11.

Abstract B23: Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides

Inhibiting Myc has long been regarded as a promising cancer treatment. However, clinical Myc inhibition was considered unfeasible due to its central role in normal proliferation and the difficulties of targeting a nuclear transcription factor. The expression of Omomyc (a Myc inhibitor derived from the dimerization and DNA-binding domain of Myc) in the KRasG12D non-small cell lung cancer (NSCLC) mouse model challenged these assumptions, as it resulted in dramatic tumor clearance with only limited and well tolerated side effects in normal tissues (Soucek et al., 2008 and 2013). Omomyc expression proved equally potent in several other mouse models of cancer, revealing the huge potential of this inhibitory approach against multiple cancer types including papilloma, pancreas and glioma (Soucek et al., 2004; Sodir et al., 2011; Annibali et al., 2014). Recently, Max*, a b-HLH-LZ peptide derived from Myc9s obligate protein partner Max, was shown to spontaneously enter cells (Montagne et al., 2012). As Omomyc and Max* display high structural homology, we hypothesized that Omomyc could also behave as a cell-penetrating peptide and thus recapitulate the effects of its transgenic counterpart. Our preliminary results show that the Omomyc peptide is well folded in solution; it transduces into cancer cells and effectively stops their proliferation in a dose-dependent manner.In vivo, nasal instillation of fluorescently-labeled Omomyc peptide leads to its rapid distribution to lungs and brain, as well as to other organs (G.I. tract, liver), as observed by IVIS® imaging and immunohistochemistry. Finally, a short treatment with the Omomyc peptide reduces the tumor size and number of Ki67 positive cells in the KRasG12D-induced NSCLC mouse model. In summary, the Omomyc cell penetrating peptide represents a new opportunity to pharmacologically inhibit Myc in a variety of malignant diseases. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Jonathan R. Whitfield, Erika Serrano del Pozo, Cynthia Tremblay, Loika Maltais, Martin Montagne, Pierre Lavigne, Laura Soucek. Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B23.

Abstract PR10: Preclinical validation of Myc inhibition by a new generation of Omomyc-based cell penetrating peptides

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e skin, lung, pancreatic cancer and glioma) and causes only mild, well-tolerated and reversible side effects in normal tissues. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which proved to be the most effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a proof of principle. Here we report the exciting finding that Omomyc-based Cell Penetrating Peptides (CPPs) are a novel, state-of-the-art method for directly utilizing Omomyc itself (and similar peptides) to treat tumors in the lung and brain, where the peptides biodistribute after intranasal administration. We provide a comprehensive preclinical validation of this innovative therapeutic approach for pharmacological inhibition of Myc in cancer cell lines of different origin and genetic make-up, as well as in a mouse model of Non-Small-Cell Lung Cancer (NSCLC), where the Omomyc-CPPs, like their transgenic counterpart before, display a dramatic therapeutic impact. Citation Format: Marie-eve Beaulieu, Jonathan Whitfield, Daniel Masso-Valles, Toni Jauset, Erika Serrano, Martin Montagne, Loika Maltais, Cynthia Tremblay, Pierre Lavigne, Laura Soucek. Preclinical validation of Myc inhibition by a new generation of Omomyc-based cell penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR10.

Abstract 2645: Preclinical validation of Myc inhibition by a new generation of Omomyc-based inhibitors

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e. skin, lung, pancreatic cancer and glioma) and causes only mild, well-tolerated and reversible side effects in normal tissues. Furthermore, we demonstrated that Myc has a non-degenerate function in cancer that cannot be replaced by other pathways, even in the most aggressive p53-null tumors. Therefore, Myc could be targeted safely and successfully without eliciting resistance to therapy. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which is an effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a successful proof of principle. Here we discuss our current research with Omomyc and our efforts to develop a clinically viable approach to Myc inhibition. One is based on the direct use of Omomyc itself as a peptide since we have discovered that it natively possesses cell-penetrating activity and it rapidly biodistributes to the lung and brain after intranasal administration. We are finding that the Omomyc peptide - like its transgenic counterpart before - has a therapeutic impact and we are continuing with the preclinical validation of this innovative therapeutic approach to pharmacological Myc inhibition. The second approach takes advantage of state-of-the-art nanocarrier technology to deliver Omomyc systemically, that can be combined with tumour-targeting ligands. These two novel Myc inhibition strategies have the potential to be translated rapidly to the clinic. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Jonathan Whitfield, Erika Serrano, Martin Montagne, Pierre Lavigne, Antonio Villaverde, Mireia Pesarrodona, Esther Vazquez, Laura Soucek. Preclinical validation of Myc inhibition by a new generation of Omomyc-based inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2645. doi:10.1158/1538-7445.AM2015-2645