David W. Woessner

Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer

Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix–loop–helix transcription factor required for hair-cell development, and its misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.

ZNO PARTICULATE MATTER REQUIRES CELL CONTACT FOR TOXICITY IN HUMAN COLON CANCER CELLS

There is ongoing concern regarding the toxicity of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. Two commercial ZnO types, one sold as a 8-10 nm powder and the other described as -325 mesh (<44 mum) powder, were evaluated in human colon-derived RKO cells. The powders had a volume-to-surface area ratio equivalent to 40 and 330 nm spheres, respectively. Both materials formed micrometer-sized agglomerates in cell culture media. The nanosized ZnO was more cytotoxic than the micrometer-sized ZnO with LC(50) values of 15 +/- 1 and 29 +/- 4 mug/cm(2), respectively. Transfer of Zn from the solid phase to the cell culture media in the presence of RKO cells was time- and concentration-dependent. However, direct particle-cell contact was required for RKO cell cytotoxicity, and the toxicity of particles was independent of the amount of soluble Zn in the cell culture media. The mechanism of cell death includes the disruption of mitochondrial function. Robust markers of apoptosis, Annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were observed when cells were treated with ZnO particulate matter but not when treated with comparable concentration of a soluble Zn salt. Both ZnO samples induced similar mechanisms of toxicity, but there was a statistically significant increase in potency per unit mass with the smaller particles.

Responses of human cells to ZnO nanoparticles: a gene transcription study

The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-α cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells.

DISRUPTION OF BCR-ABL COILED-COIL OLIGOMERIZATION BY DESIGN*

Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

Development of an effective therapy for chronic myelogenous leukemia.

AbstractTargeted small-molecule drugs have revolutionized treatment of chronic myeloid leukemia (CML) during the last decade. These agents interrupt a constitutively active BCR-ABL, the causative agent for CML, by interfering with adenosine 5′ triphosphate–dependent ABL tyrosine kinase. Although the efficacy of tyrosine kinase inhibitors (TKIs) has resulted in overall survival of greater than 90%, TKIs are not curative. Moreover, no currently approved TKIs are effective against the T315I BCR-ABL variant. However, a new generation of TKIs with activity against T315I is on the horizon. We will highlight the clinical utility of historical CML therapeutics, those used today (first- and second-generation TKIs), and discuss treatment modalities that are under development. Recent advances have illuminated the complexity of CML, especially within the marrow microenvironment. We contend that the key to curing CML will involve strategies beyond targeting BCR-ABL because primitive human CML stem cells are not dependent on BCR-ABL. Ultimately, drug combinations or exploiting synthetic lethality may transform responses into definitive cures for CML.

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

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.

Multidomain targeting of Bcr-Abl by disruption of oligomerization and tyrosine kinase inhibition: toward eradication of CML.

The oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain to function [Bartram, C. R.; et al. Nature 1983, 306 (5940), 277-280; and Zhao, X.; et al. Nat. Struct. Biol. 2002, 9(2), 117-120]. While tyrosine kinase inhibitors (TKIs) have shown great efficacy as treatment options for CML, their use may cause an acquisition of mutations in the tyrosine kinase domain, which prevent TKI binding and lead to a loss in activity [Woessner, D. W.; et al. Cancer J. 2011, 17(6), 477-486]. Previously, we have shown that a rationally modified coiled-coil domain (CC(mut3)) can disrupt this oligomerization, inhibit proliferation, and induce apoptosis in CML cells [Dixon, A. S.; et al. Mol. Pharmaceutics 2012, 9(1), 187-195]. Here, we show that using the most recently approved TKI, ponatinib (Iclusig), in combination with CC(mut3) allows a dose reduction of ponatinib and increased therapeutic efficacy in vitro measured by reduction in kinase activity, induction of apoptosis via caspase-3/7 and 7-AAD/Annexin V assays, and reduced transformative ability measured by a colony forming assay. The combination was effective not only in cells containing wild-type Bcr-Abl (K562, Ba/F3-p210) but also cells with Bcr-Abl containing the T315I mutation (Ba/F3-p210-T315I). In addition, we report for the first time the ability of CC(mut3) alone to inhibit the T315I mutant form of Bcr-Abl. This novel combination may prove to be more potent than single agent therapies and should be further explored for clinical use.

Disrupting BCR-ABL in combination with secondary leukemia-specific pathways in CML cells leads to enhanced apoptosis and decreased proliferation.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell. Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which block ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use is required in order to treat CML. The primary failure for TKIs is through the development of a resistant population due to mutations in the TKI binding regions. This led us to develop the mutant coiled-coil, CC(mut2), an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation. In this article, we explore additional pathways that are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach, we test the combination of CC(mut2) and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox5 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-1 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy using chloroquine in addition to blocking BCR-ABL signaling with the CC(mut2) was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways.

A coiled-coil mimetic intercepts BCR-ABL1 dimerization in native and kinase-mutant chronic myeloid leukemia

Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CCmut3, which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CCmut3 exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CCmut3 inhibits colony formation by primary CML CD34+ cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.