Hanshi Sun

Association Between Imatinib-Resistant BCR-ABL Mutation-Negative Leukemia and Persistent Activation of LYN Kinase

BACKGROUND Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines. METHODS We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state-specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells. RESULTS Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease. CONCLUSIONS LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.

Bcr-Abl ubiquitination and Usp9x inhibition block kinase signaling and promote CML cell apoptosis

Although chronic myelogenous leukemia (CML) is effectively controlled by Bcr-Abl kinase inhibitors, resistance to inhibitors, progressive disease, and incomplete eradication of Bcr-Abl-expressing cells are concerns for the long-term control and suppression of this disease. We describe a novel approach to targeting key proteins in CML cells with a ubiquitin-cycle inhibitor, WP1130. Bcr-Abl is rapidly modified with K63-linked ubiquitin polymers in WP1130-treated CML cells, resulting in its accumulation in aggresomes, where is it unable to conduct signal transduction. Induction of apoptosis because of aggresomal compartmentalization of Bcr-Abl was observed in both imatinib-sensitive and -resistant cells. WP1130, but not Bcr-Abl kinase inhibitors, directly inhibits Usp9x deubiquitinase activity, resulting in the down-regulation of the prosurvival protein Mcl-1 and facilitating apoptosis. These results demonstrate that ubiquitin-cycle inhibition represents a novel and effective approach to blocking Bcr-Abl kinase signaling and reducing Mcl-1 levels to engage CML cell apoptosis. This approach may be a therapeutic option for kinase inhibitor-resistant CML patients.

Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy

Significance Mutations within the BCR-ABL1 kinase domain lead to resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia. We show a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKIs. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs unveiled the basis of TKI resistance. Modeling of BCR-ABL1 in complex with ponatinib, a potent pan-BCR-ABL1 TKI, highlighted the presence of complex BCR-ABL1 mutant proteins capable of escaping all available TKI therapy. The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia. Sensitive sequencing techniques have revealed a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKI therapy. To investigate the molecular consequences of such complex mutant proteins with regards to TKI resistance, we determined by cloning techniques the presence of polymutants in a cohort of chronic-phase patients receiving imatinib followed by dasatinib therapy. The analysis revealed a high frequency of polymutant BCR-ABL1 alleles even after failure of frontline imatinib, and also the progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of sequential TKI therapy. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs revealed the basis of TKI resistance. Modeling of BCR-ABL1 in complex with the potent pan-BCR-ABL1 TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling unique mechanisms of escape to ponatinib therapy.

Targeting deubiquitinase activity with a novel small-molecule inhibitor as therapy for B-cell malignancies

Usp9x was recently shown to be highly expressed in myeloma patients with short progression-free survival and is proposed to enhance stability of the survival protein Mcl-1. In this study, we found that the partially selective Usp9x deubiquitinase inhibitor WP1130 induced apoptosis and reduced Mcl-1 protein levels. However, short hairpin RNA-mediated knockdown (KD) of Usp9x in myeloma cells resulted in transient induction of apoptosis, followed by a sustained reduction in cell growth. A compensatory upregulation of Usp24, a deubiquitinase closely related to Usp9x, in Usp9x KD cells was noted. Direct Usp24 KD resulted in marked induction of myeloma cell death that was associated with a reduction of Mcl-1. Usp24 was found to sustain myeloma cell survival and Mcl-1 regulation in the absence of Usp9x. Both Usp9x and Usp24 were expressed and activated in primary myeloma cells whereas Usp24 protein overexpression was noted in some patients with drug-refractory myeloma and other B-cell malignancies. Furthermore, we improved the drug-like properties of WP1130 and demonstrated that the novel compound EOAI3402143 dose-dependently inhibited Usp9x and Usp24 activity, increased tumor cell apoptosis, and fully blocked or regressed myeloma tumors in mice. We conclude that small-molecule Usp9x/Usp24 inhibitors may have therapeutic activity in myeloma.

p53 stabilization induces apoptosis in chronic myeloid leukemia blast crisis cells

Philadelphia chromosome positive chronic myeloid leukemia has a progressive course starting in a benign phase and terminating in a blastic phase. In this study, we show that human homolog double minute 2 (HDM2) inhibition, with MI-219—a novel compound, and consequently p53 stabilization induce chronic myeloid leukemia (CML) blast crisis cells to undergo apoptosis regardless of the presence of the T315I mutation in the BCR–ABL kinase domain. The response to MI-219 is associated with the downregulation of c-Myc and the induction of p21WAF1. The p53 target and pro-apoptotic proteins PUMA, Noxa and Bax are induced, whereas full length Bid protein decreases with increased activity of pro-apoptotic cleaved Bid, and decrease of Mcl-1 is observed by increased caspase activity. CD95/FAS (FAS antigen) receptor is also induced by MI-219, indicating that both intrinsic and extrinsic apoptotic responses are transcriptionally induced. In addition, p53 protein accumulates in the mitochondrial fraction of treated cells involved in transcription-independent induction of apoptosis. We conclude that HDM-2 inhibition with MI-219 effectively induces p53-dependent apoptosis in most blast crisis CML cells, with or without BCR–ABL mutation(s).

Targets and effectors of the cellular response to aurora kinase inhibitor MK-0457 (VX-680) in imatinib sensitive and resistant chronic myelogenous leukemia

MK-0457 inhibits aurora, BCR-ABL and other kinases and may be clinically active in imatinib resistant leukemia. To define mediators of MK-0457 responsiveness, kinase inhibitory profiles were examined in multiple cell models of imatinib sensitive and resistant disease. Aurora and BCR-ABL kinase inhibition were consistently measured at 20-100 nM and 2-10 microM MK-0457, respectively, but expression of T315I-BCR-ABL and overexpression of Lyn kinase reduced MK-0457 sensitivity. Aurora kinase inhibition was associated with cell cycle restriction and p53 induction and p53-null cells were far less responsive to MK-0457, requiring BCR-ABL inhibitory concentrations for apoptotic activity. In wild-type p53 expressing CML cells MK-0457 sensitivity was modulation by alterations in p53 levels through HDM-2 inhibition and gene silencing. MK-0457 suppressed aurora kinase activity and induced apoptosis in imatinib resistant clinical specimens expressing T315I and other BCR-ABL mutations without effecting BCR-ABL kinase activity. Together, these results suggest that MK-0457 apoptotic activity in CML cells is primarily associated with aurora kinase inhibition but can be altered by multiple molecular changes associated with disease progression or acquisition of imatinib resistance.

Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma

ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications.

Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic β-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote β-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat β cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

Anti-infective activity of 2-cyano-3-acrylamide inhibitors with improved drug-like properties against two intracellular pathogens

ABSTRACT Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the hosts ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes. We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.

Abstract 4435: Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Melanoma is an aggressive skin cancer characterized by gene mutations, high metastatic potential and few clinically effective systemic therapies. BRAF, MEK1 kinase inhibitors and CTLA4 antagonist have improved patient survival, but only a subset of patients respond or derived long-lasting clinical benefit, illustrating the need for discovery of additional targets and pathways that may be inhibited with small molecules. Given the clinical impact of inhibitors of the ubiquitin-proteasome system in other cancers, we explored the potential for aberrant deubiquitinase (DUB) activity to play a role in the tumorigenic properties of melanoma. We also assessed whether specific DUBs play a role in apoptotic responsiveness and resistance to kinase inhibitors. Initially, we noted that the activity of several DUBs, including Usp9x and Usp5, was elevated in melanoma compared to normal melanocytes. Further, Usp9x and Usp5 expression and activity were up-regulated by expression of BRAFV600E in 293T cells and kinase inhibition partially suppressed both Usp9x and Usp5 activity in vemurafenib sensitive, but not resistant cells. Usp9x knockdown (KD) in BRAF mutant or wild-type melanoma did not affect 2-dimensional cell growth or survival but significantly amplified the apoptotic activity of BRAF or MEK1 inhibitors, respectively. Usp5 KD in melanoma suppressed cell growth by reinforced the S/G2-M checkpoint, enhanced extrinsic caspase activation through modulation of p53 and FAS levels and amplified the apoptotic activity of kinase inhibitors (and other chemotherapeutic agents) and overcame vemurafenib resistance in A375R cells. To further assess the clinical relevance of activated DUBs in melanoma, primary human melanoma explants characterized for their metastatic efficiency in patients and NSG mice demonstrated that Usp9x and Usp5 expression and activity were elevated in efficient metastasizers, but only a subset of inefficient metastasizers, suggesting a potential role for DUBs in the regulation of metastatic potential. To investigate that possibly, control and Usp9x KD cells were grown in Matrigel or suspension culture and colony growth and survival, as well as Usp9x activity were examined. Usp9x KD significantly suppressed melanoma colony growth and fully reduced survival of highly metastatic SK-Mel147 cells in 3D culture. Growth in 3D conditions resulted in activation of Usp9x, but also increased their sensitivity (IC50 <400 nM) to our partially selective Usp9x inhibitor, EOAI3402143 (G9). G9 inhibits both Usp9x and Usp5 activity and phenocopied the activities of both Usp9x and Usp5 KD cells. G9 was safely administered to NSG mice, with complete block of melanoma growth. We conclude that targeted inhibition of specific DUBs will suppress metastatic melanoma and amplify existing melanoma therapies. Citation Format: Harish Potu, Anupama Pal, Hanshi Sun, Luke Peterson, Moshe Talpaz, Monique Verhaegen, Juxiang Cao, Ugur Eskiocak, Sean Morrison, Nicholas J. Donato. Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2014-4435