Karina J. Matissek

The DNA Binding Domain of p53 Is Sufficient To Trigger a Potent Apoptotic Response at the Mitochondria

The tumor suppressor p53 is one of the most studied proteins in human cancer.1-3 While nuclear p53 has been utilized for cancer gene therapy, mitochondrial targeting of p53 has not been fully exploited to date.4,5 In response to cellular stress, p53 translocates to the mitochondria and directly interacts with Bcl-2 family proteins including antiapoptotic Bcl-XL and Bcl-2 and proapoptotic Bak and Bax.6 Antiapoptotic Bcl-XL forms inhibitory complexes with proapoptotic Bak and Bax preventing their homo-oligomerization.7 Upon translocation to the mitochondria, p53 binds to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances their homo-oligomerization.8 Bak and Bax homotetramer formation disrupts the mitochondrial outer membrane, releases antiapoptotic factors such as cytochrome c and triggers a rapid apoptotic response mediated by caspase induction.9 It is still unclear if the MDM2 binding domain (MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD) of p53 are the domains responsible for interaction with Bcl-XL.10-17 The purpose of this work is to determine if a smaller functional domain of p53 is capable of inducing apoptosis similarly to full length p53. To explore this question, different domains of p53 (MBD, PRD, DBD) were fused to the mitochondrial targeting signal (MTS) from Bcl-XL to ensure Bcl-XL specific targeting.18 The designed constructs were tested for apoptotic activity (TUNEL, Annexin-V, and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231), in a cervical cancer cell line (HeLa) and in non-small cell lung adenocarcinoma cells H1373. Our results indicate that DBD-XL (p53 DBD fused to the Bcl-XL MTS) reproduces (in T47D cells) or demonstrates increased apoptotic activity (in MCF-7, MDA-MB-231, and HeLa cells) compared to p53-XL (full length p53 fused to Bcl-XL MTS). Additionally, mitochondrial dependent apoptosis assays (TMRE, caspase-9), co-IP and overexpression of Bcl-XL in T47D cells suggest that DBD fused to XL MTS may bind to and inhibit Bcl-XL. Taken together, our data demonstrates for the first time that the DBD of p53 may be the minimally necessary domain for achieving apoptosis at the mitochondria in multiple cell lines. This work highlights the role of small functional domains of p53 as a novel cancer biologic therapy.

A Chimeric p53 Evades Mutant p53 Transdominant Inhibition in Cancer Cells

Because of the dominant negative effect of mutant p53, there has been limited success with wild-type (wt) p53 cancer gene therapy. Therefore, an alternative oligomerization domain for p53 was investigated to enhance the utility of p53 for gene therapy. The tetramerization domain of p53 was substituted with the coiled-coil (CC) domain from Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains proper nuclear localization in breast cancer cells detected via fluorescence microscopy and shows a similar expression profile of p53 target genes as wt-p53. Additionally, similar tumor suppressor activities of p53-CC and wt-p53 were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD), and colony-forming assays. Furthermore, p53-CC was found to cause apoptosis in four different cancer cell lines, regardless of endogenous p53 status. Interestingly, the transcriptional activity of p53-CC was higher than wt-p53 in 3 different reporter gene assays. We hypothesized that the higher transcriptional activity of p53-CC over wt-p53 was due to the sequestration of wt-p53 by endogenous mutant p53 found in cancer cells. Co-immunoprecipitation revealed that wt-p53 does indeed interact with endogenous mutant p53 via its tetramerization domain, while p53-CC escapes this interaction. Therefore, we investigated the impact of the presence of a transdominant mutant p53 on tumor suppressor activities of wt-p53 and p53-CC. Overexpression of a potent mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53, p53-CC retains the same level of tumor suppressor activity. Finally, viral transduction of wt-p53 and p53-CC into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53.

Enhanced and Selective Killing of Chronic Myelogenous Leukemia Cells with an Engineered BCR-ABL Binding Protein and Imatinib

The oncoprotein Bcr-Abl stimulates prosurvival pathways and suppresses apoptosis from its exclusively cytoplasmic locale, but when targeted to the mitochondrial compartment of leukemia cells, Bcr-Abl was potently cytotoxic. Therefore, we designed a protein construct to act as a mitochondrial chaperone to move Bcr-Abl to the mitochondria. The chaperone (i.e., the 43.6 kDa intracellular cryptic escort (iCE)) contains an EGFP tag and two previously characterized motifs: (1) an optimized Bcr-Abl binding motif that interacts with the coiled-coil domain of Bcr (ccmut3; 72 residues), and (2) a cryptic mitochondrial targeting signal (cMTS; 51 residues) that selectively targets the mitochondria in oxidatively stressed cells (i.e., Bcr-Abl positive leukemic cells) via phosphorylation at a key residue (T193) by protein kinase C. While the iCE colocalized with Bcr-Abl, it did not relocalize to the mitochondria. However, the iCE was selectively toxic to Bcr-Abl positive K562 cells as compared to Bcr-Abl negative Cos-7 fibroblasts and 1471.1 murine breast cancer cells. The toxicity of the iCE to leukemic cells was equivalent to 10 μM imatinib at 48 h and the iCE combined with imatinib potentiated cell death beyond imatinib or the iCE alone. Substitution of either the ccmut3 or the cMTS with another Bcr-Abl binding domain (derived from Ras/Rab interaction protein 1 (RIN1; 295 residues)) or MTS (i.e., the canonical IMS derived from Smac/Diablo; 49 residues) did not match the cytotoxicity of the iCE. Additionally, a phosphorylation null mutant of the iCE also abolished the killing effect. The mitochondrial toxicity of Bcr-Abl and the iCE in Bcr-Abl positive K562 leukemia cells was confirmed by flow cytometric analysis of 7-AAD, TUNEL, and annexin-V staining. DNA segmentation and cell viability were assessed by microscopy. Subcellular localization of constructs was determined using confocal microscopy (including statistical colocalization analysis). Overall, the iCE was highly active against K562 leukemia cells and the killing effect was dependent upon both the ccmut3 and functional cMTS domains.

Delivery of a monomeric p53 subdomain with mitochondrial targeting signals from pro-apoptotic Bak or Bax.

Purposep53 targeted to the mitochondria is the fastest and most direct pathway for executing p53 death signaling. The purpose of this work was to determine if mitochondrial targeting signals (MTSs) from pro-apoptotic Bak and Bax are capable of targeting p53 to the mitochondria and inducing rapid apoptosis.Methodsp53 and its DNA-binding domain (DBD) were fused to MTSs from Bak (p53-BakMTS, DBD-BakMTS) or Bax (p53-BaxMTS, DBD-BaxMTS). Mitochondrial localization was tested via fluorescence microscopy in 1471.1 cells, and apoptosis was detected via 7-AAD in breast (T47D), non-small cell lung (H1373), ovarian (SKOV-3) and cervical (HeLa) cancer cells. To determine that apoptosis is via the intrinsic apoptotic pathway, TMRE and caspase-9 assays were conducted. Finally, the involvement of p53/Bak specific pathway was tested.ResultsMTSs from Bak and Bax are capable of targeting p53 to the mitochondria, and p53-BakMTS and p53-BaxMTS cause apoptosis through the intrinsic apoptotic pathway. Additionally, p53-BakMTS, DBD-BakMTS, p53-BaxMTS and DBD-BaxMTS caused apoptosis in T47D, H1373, SKOV-3 and HeLa cells. The apoptotic mechanism of p53-BakMTS and DBD-BakMTS was Bak dependent.ConclusionOur data demonstrates that p53-BakMTS (or BaxMTS) and DBD-BakMTS (or BaxMTS) cause apoptosis at the mitochondria and can be used as a potential gene therapeutic in cancer.

Re-engineered p53 chimera with enhanced homo-oligomerization that maintains tumor suppressor activity.

The use of the tumor suppressor p53 for gene therapy of cancer is limited by the dominant negative inactivating effect of mutant endogenous p53 in cancer cells. We have shown previously that swapping the tetramerization domain (TD) of p53 with the coiled-coil (CC) from Bcr allows for our chimeric p53 (p53-CC) to evade hetero-oligomerization with endogenous mutant p53. This enhances the utility of this construct, p53-CC, for cancer gene therapy. Because domain swapping to create p53-CC could result in p53-CC interacting with endogenous Bcr, which is ubiquitous in cells, modifications on the CC domain are necessary to minimize potential interactions with Bcr. Hence, we investigated the possible design of mutations that will improve homodimerization of CC mutants and disfavor hetero-oligomerization with wild-type CC (CCwt), with the goal of minimizing potential interactions with endogenous Bcr in cells. This involved integrated computational and experimental approaches to rationally design an enhanced version of our chimeric p53-CC tumor suppressor. Indeed, the resulting lead candidate p53-CCmutE34K-R55E avoids binding to endogenous Bcr and retains p53 tumor suppressor activity. Specifically, p53-CCmutE34K-R55E exhibits potent apoptotic activity in a variety of cancer cell lines, regardless of p53 status (in cells with mutant p53, wild-type p53, or p53-null cells). This construct overcomes the dominant negative effect limitation of wt p53 and has high significance for future gene therapy for treatment of cancers characterized by p53 dysfunction, which represent over half of all human cancers.

Expressed Gene Fusions as Frequent Drivers of Poor Outcomes in Hormone Receptor–Positive Breast Cancer

We sought to uncover genetic drivers of hormone receptor-positive (HR+) breast cancer, using a targeted next-generation sequencing approach for detecting expressed gene rearrangements without prior knowledge of the fusion partners. We identified intergenic fusions involving driver genes, including PIK3CA, AKT3, RAF1, and ESR1, in 14% (24/173) of unselected patients with advanced HR+ breast cancer. FISH confirmed the corresponding chromosomal rearrangements in both primary and metastatic tumors. Expression of novel kinase fusions in nontransformed cells deregulates phosphoprotein signaling, cell proliferation, and survival in three-dimensional culture, whereas expression in HR+ breast cancer models modulates estrogen-dependent growth and confers hormonal therapy resistance in vitro and in vivo Strikingly, shorter overall survival was observed in patients with rearrangement-positive versus rearrangement-negative tumors. Correspondingly, fusions were uncommon (<5%) among 300 patients presenting with primary HR+ breast cancer. Collectively, our findings identify expressed gene fusions as frequent and potentially actionable drivers in HR+ breast cancer.Significance: By using a powerful clinical molecular diagnostic assay, we identified expressed intergenic fusions as frequent contributors to treatment resistance and poor survival in advanced HR+ breast cancer. The prevalence and biological and prognostic significance of these alterations suggests that their detection may alter clinical management and bring to light new therapeutic opportunities. Cancer Discov; 8(3); 336-53. ©2017 AACR.See related commentary by Natrajan et al., p. 272See related article by Liu et al., p. 354This article is highlighted in the In This Issue feature, p. 253.

Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model

Inactivation of p53 pathway is reported in more than half of all human tumors and can be correlated to malignant development. Missense mutation in the DNA binding region of p53 is the most common mechanism of p53 inactivation in cancer cells. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain. Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have previously designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain capable of escaping transdominant inhibition by mutant p53 in vitro. In this report, we demonstrate the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model in vivo. In addition, we illustrate the profound effects of the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells. Finally, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model.

Abstract 790: An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The tumor suppressor p53 is mutated in more than 50% of all cancers. Our data have shown that swapping the tetramerization domain (TD) of p53 with an alternative oligomerization domain enhances the utility of p53 for cancer gene therapy. Previously, we have swapped the tetramerization domain of p53 with the coiled-coil (CC) domain from breakpoint cluster region (Bcr). This alteration of the oligomerization motif of the tumor suppressor allowed for our construct, namely p53-CC, to evade hetero-oligomerization with endogenous mutant p53 commonly found in cancer cells. This proves to be critical since mutant p53 has a transdominant inhibitory effect over wild-type p53 upon hetero-oligomerization. A co-immunopreciptation (co-IP) experiment in human breast ductal carcinoma (T47D) cells validated our hypothesis that endogenous p53 interacts directly with exogenous wild-type p53, which is due to hetero-oligomerization via their TDs. In contrast, p53-CC, which lacks the TD, evaded binding to endogenous p53. Furthermore, overexpression of a potent mutant p53 (contains three hotspot mutations; R175H, R248W, and R273H) significantly impaired the function of exogenous wild-type p53, while the tumor suppressor activity of p53-CC was not affected. To further increase the apoptotic potential of p53-CC, rational design of mutations in the CC domain were investigated for two purposes: first to increase the dimerization affinity of CC, and second to prevent any potential interaction with endogenous Bcr. Leucine at residue 62 of the CC domain forms a critical hydrophobic pocket at the dimeric interface. Therefore, mutating this residue into the more hydrophobic isoleucine (with similar molecular weight),, should increase CC dimerization affinity. Results from a standard mammalian two-hybrid assay will be presented to validate the binding affinity of our mutant CC compared to the native CC. Furthermore, two additional mutations on the CC domain (R55E and E34K) were carried out to prevent any potential interaction of our construct with endogenous Bcr. These two mutations introduce a salt bridge that promotes homo-dimerization of mutant CC as well as preventing hetero-dimerization with native CC representative of endogenous Bcr. This will be confirmed using another co-IP experiment that shows no interaction between mutant CC and endogenous Bcr. Finally, two apoptotic assays were carried out to verify that introducing the aforementioned mutations did not cause any loss of tumor suppressor activity of our enhanced p53-CC variant. In summary, a p53 with altered TD has been designed for exclusive homo-dimerization and has potent activity as a tumor suppressor. Our ultimate goal is to use this version of p53 as a new gene therapeutic, capable of bypassing the dominant negative effect in cancers with mutated or mislocalized p53. Citation Format: Abood Okal, Mohanad Mossalam, Karina J. Matissek, Carol S. Lim. An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2013-790

Abstract 791: Targeting small domains of p53 to mitochondrial Bcl-XL for cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The tumor suppressor p53 is involved in transcriptional induction or repression of various genes resulting in cell cycle arrest, senescence, DNA repair and apoptosis. While most of these functions can be linked to its role as a transcription factor in the nucleus, p53 also triggers apoptosis via the mitochondrial pathway. In response to cellular stress, p53 translocates to the mitochondria and directly interacts with Bcl-2 protein family members including anti-apoptotic Bcl-XL and Bcl-2 and pro-apoptotic Bak and Bax. Anti-apoptotic Bcl-XL forms inhibitory complexes with pro-apoptotic Bak and Bax preventing their homo-oligomerization. Upon translocation to the mitochondria p53 binds to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances their homo-oligomerization. Bak and Bax homo-tetramer formation disrupts the mitochondrial outer membrane, releases anti-apoptotic factors such as cytochrome c and triggers a rapid apoptotic response mediated by caspase induction. There are conflicting reports suggesting that the MDM2 binding domain (MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD) of p53 are the domains for interacting to Bcl-XL. The purpose of this work is to determine which subdomain of p53 binds optimally to Bcl-XL while maintaining the full apoptotic activity of full length p53. Different domains of p53 (MBD, PRD, DBD) were fused to the mitochondrial targeting signal from Bcl-XL to ensure Bcl-XL specific targeting. The designed constructs were tested for apoptotic activity (TUNEL, Annexin-V, and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231), in a cervical cancer cell line (HeLa), and in human leukemic cells (K562). Our results indicate that DBD-XL (p53 DBD fused to the Bcl-XL MTS) shows the same (in T47D and, K562 cells) or higher (in MCF-7, MDA-MB-231, and HeLa cells) apoptotic activity compared to p53-XL. Additionally, over-expression of Bcl-XL in T47D cells confirmed that DBD directly binds and inhibits Bcl-XL. Further, binding affinity of designed constructs to Bcl-XL will be explored via co-immunoprecipitation (co-IP) and mammalian two-hybrid assay. Taken together, our data highlights that DBD can be used instead of full length p53 for achieving apoptosis at the mitochondria. The benefit of decreasing the overall size of p53 while maintaining full apoptotic activity allows for better drug delivery options. Furthermore, DBD-XL-mediated apoptosis of multiple cell lines including such as breast cancer cells, cervical carcinoma cells, and leukemia cells highlighting the role of p53 as the ultimate target for cancer therapy. Citation Format: Karina J. Matissek, Mohanad Mossalam, Abood Okal, Carol S. Lim. Targeting small domains of p53 to mitochondrial Bcl-XL for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2013-791

Abstract 1176: Bypassing the dominant-negative effect of mutant p53 in cancer cells

The tumor suppressor p53 is mutated in more than 50% of all cancers. The transcriptional activity of p53 depends on localization to the nucleus and formation of p53 tetramers. Use of wild-type (wt) p53 for gene therapy is limited due to the dominant-negative effect of mutant (mut) p53 over wt p53. Mut p53 will hetero-oligomerize with wt p53 via the tetramerization domain (TD) found in p53, deactivating the tumor suppressor function of wt p53. In order to use p53 for gene therapy, an alternative oligomerization domain for p53 was investigated. The coiled-coil (CC) domain from Bcr (breakpoint cluster region), similar to the native TD of p53, can form an antiparallel dimer of dimers. The TD from p53 was then substituted with this CC, and the activity of this new p53 (p53-CC) was tested. We evaluated the transcriptional activity of p53-CC compared to wt p53 in a dual luciferase reporter gene assay. To demonstrate the ability of our p53-CC to bypass the dominant-negative effect prompted by hetero-oligomers, the reporter gene assay was carried in human breast adenocarcinoma (MCF-7) and human breast ductal carcinoma (T47D) cell lines. MCF-7 cells contain wt p53 population that is mislocalized to the cytoplasm, whereas, T47D cells contain mutant p53. Indeed, data from the reporter gene experiment shows a reduction in wt p53 activity introduced to T47D cells compared to MCF-7 cells, while the activity of p53-CC remains consistent regardless of the presence of an endogenous mut p53 population. We also tested the biological activity of p53-CC and its ability to induce apoptosis using 7-AAD and Annexin-V assays (late stage apoptosis). In addition, Caspase-3/7 assay (mid-stage apoptosis) was performed to further validate a p53-dependent apoptotic pathway. Data obtained from the aforementioned experiments suggest that p53-CC has a very similar biological activity to wt p53. Furthermore, we examined the gene expression profile of p53-CC using wt p53 as a reference. The assay was performed using real-time PCR array technology to analyze the expression of 84 genes related to p53-mediated signal transduction. Two negative controls were used; the first is a p53 construct that lacks its tetramerization domain (p53ΔTDC) and should not possess any transcriptional activity, and the second is the CC by itself. Indeed, p53-CC shows comparable gene expression profile to wt p53 and has the ability to transactivate the various p53 target genes. In summary, p53-CC retains p53 activity and excludes hetero-oligomer formation, overcoming a major barrier in using p53 for cancer gene therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1176. doi:1538-7445.AM2012-1176