Proteolysis-Targeting Chimeras (PROTACs) targeting the BCR-ABL for the treatment of chronic myeloid leukemia - a patent review.

INTRODUCTION: PROteolysis-TArgeting Chimeras (PROTACs) allow the selective degradation of a protein of interest (POI) by the ubiquitin-proteasome system (UPS). With this unique mechanism of action, the research and development of PROTACs that target the Breakpoint Cluster Region Abelson (BCR-ABL) tyrosine kinase (TK) has been increasing dramatically, as they are promising molecules in the treatment of Chronic Myeloid Leukemia (CML), one of the main hematological malignancies, which results from an uncontrolled myeloproliferation due to the constitutive activation of BCR-ABL. AREAS COVERED: This review summarizes the patents/applications published in the online databases like Espacenet or World Intellectual Property Organization regarding PROTACs that promote BCR-ABL degradation. Patents will be described mostly in terms of chemical structure, biochemical/pharmacological activities, and potential clinical applications. EXPERT OPINION: The recent discovery of the enormous potential of PROTACs led to the creation of new compounds capable of degrading BCR-ABL for the treatment of CML. Although still in reduced numbers, and in the pre-clinical phase of development, some compounds have already been shown to overcome some of the difficulties presented by conventional BCR-ABL inhibitors, such as the well-known imatinib. Therefore, it is very likely that some of the present PROTACs will enter future CML therapy in the coming years.

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire.

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer-dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer-dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.

The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia.

BCR-ABL drives chronic myeloid leukemia (CML). BCR binding to GRB2 transduces signaling via the Ras/MAPK pathway. Despite considerable data confirming the binding, molecular-level understanding of exactly how the two proteins interact, and, especially, what are the determinants of the specificity of the SH2GRB2 domain-phosphorylated BCR (pBCR) recognition are still open questions. Yet, this is vastly important for understanding binding selectivity, and for predicting the phosphorylated receptors, or peptides, that are likely to bind. Here, we uncover these determinants and ascertain to what extent they relate to the affinity of the interaction. Toward this end, we modeled the complexes of the pBCR and SH2GRB2 and other pY/Y-peptide-SH2 complexes and compared their specificity and affinity. We observed that pBCR's 176FpYVNV180 motif is favorable and specific to SH2GRB2, similar to pEGFR, but not other complexes. SH2GRB2 contains two binding pockets: pY-binding recognition pocket triggers binding, and the specificity pocket whose interaction is governed by N179 in pBCR and W121 in SH2GRB2. Our proposed motif with optimal affinity to SH2GRB2 is E/D-pY-E/V-N-I/L. Collectively, we provide the structural basis of BCR-ABL recruitment of GRB2, outline its specificity hallmarks, and delineate a blueprint for prediction of BCR-binding scaffolds and for therapeutic peptide design.

Investigation on the interaction behavior of afatinib, dasatinib, and imatinib docked to the BCR-ABL protein.

Chronic myeloid leukemia (CML) is a pathological condition associated with the uncontrolled proliferation of white blood cells and respective loss of function. Imatinib was the first drug that could effectively treat this condition, but its use is hindered by the development of mutations of the BCR-ABL protein, which are the cause of resistance. Therefore, dasatinib and afatinib present similarities that can be explored to discover new molecules capable of overcoming the effects of imatinib. Afatinib exhibited electronic and docking behavior, indicating that a replacement with some minor modifications could design a new potential inhibitor. The amide group in each candidate is clearly of pharmacophoric importance, and it needs to concentrate a negative region. Sulfur group presents a good pharmacophoric profile, which was shown by dasatinib results, adding to the influence of the Met318 residue in the target protein active site configuration. This behavior suggests that the sulfur atom and other fragments that have an affinity for the methionine sidechain may provide a significant positive effect when present in TKI molecules such as afatinib or dasatinib.

Inhibition of AKR1B10-mediated metabolism of daunorubicin as a novel off-target effect for the Bcr-Abl tyrosine kinase inhibitor dasatinib.

Bcr-Abl tyrosine kinase inhibitors significantly improved Philadelphia chromosome-positive leukaemia therapy. Apart from Bcr-Abl kinase, imatinib, dasatinib, nilotinib, bosutinib and ponatinib are known to have additional off-target effects that might contribute to their antitumoural activities. In our study, we identified aldo-keto reductase 1B10 (AKR1B10) as a novel target for dasatinib. The enzyme AKR1B10 is upregulated in several cancers and influences the metabolism of chemotherapy drugs, including anthracyclines. AKR1B10 reduces anthracyclines to alcohol metabolites that show less antineoplastic properties and tend to accumulate in cardiac tissue. In our experiments, clinically achievable concentrations of dasatinib selectively inhibited AKR1B10 both in experiments with recombinant enzyme (Ki = 0.6 µM) and in a cellular model (IC50 = 0.5 µM). Subsequently, the ability of dasatinib to attenuate AKR1B10-mediated daunorubicin (Daun) resistance was determined in AKR1B10-overexpressing cells. We have demonstrated that dasatinib can synergize with Daun in human cancer cells and enhance its therapeutic effectiveness. Taken together, our results provide new information on how dasatinib may act beyond targeting Bcr-Abl kinase, which may help to design new chemotherapy regimens, including those with anthracyclines.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

OBJECTIVE: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. METHODOLOGY: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling  studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. RESULTS: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. CONCLUSION: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second  generation and third generation TKIs.

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.

Induction of CML-specific immune response through cross-presentation triggered by CTP-mediated BCR-ABL-derived peptides.

Although targeted therapy using tyrosine kinase inhibitors (TKIs) has made remarkable progress in treating chronic myeloid leukemia (CML), this disease remains largely incurable, warranting further investigation of new therapeutic strategies. BCR-ABL is a highly specific tumor antigen in CML and provides an attractive opportunity for vaccination therapy. Exogenous antigens must be presented on MHC class I molecules-via a process termed cross-presentation-to activate specific cytotoxic T lymphocyte response. The relative efficiency of cross-presentation is determined in part by the ability of dendritic cells (DCs) to internalize and present antigens. Here, we present a novel tool that uses cytoplasmic transduction peptide (CTP) to facilitate the internalization of antigens by DCs in an endocytosis-independent manner, which greatly enhances the efficiency of antigen presentation, thereby inducing stronger cytotoxic activity to ensure the elimination of CML cells. The data suggest that CTP-fused CML-specific peptides can be applied in vaccination therapies for CML patients.

A combined computational and experimental strategy identifies mutations conferring resistance to drugs targeting the BCR-ABL fusion protein.

Drug resistance is of increasing concern, especially during the treatments of infectious diseases and cancer. To accelerate the drug discovery process in combating issues of drug resistance, here we developed a computational and experimental strategy to predict drug resistance mutations. Using BCR-ABL as a case study, we successfully recaptured the clinically observed mutations that confer resistance imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. We then experimentally tested the predicted mutants in vitro. We found that although all mutants showed weakened binding strength as expected, the binding constants alone were not a good indicator of drug resistance. Instead, the half-maximal inhibitory concentration (IC50) was shown to be a good indicator of the incidence of the predicted mutations, together with change in catalytic efficacy. Our suggested strategy for predicting drug-resistance mutations includes the computational prediction and in vitro selection of mutants with increased IC50 values beyond the drug safety window.

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study.

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph + ALL). Ph + ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph + ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph + ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.

Elucidation of protein interactions necessary for the maintenance of the BCR-ABL signaling complex.

Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.

Homoharringtonine promotes BCR­ABL degradation through the p62­mediated autophagy pathway.

Drug resistance to tyrosine kinase inhibitors (TKIs) is currently a clinical problem in patients with chronic myelogenous leukemia (CML). Homoharringtonine (HHT) is an approved treatment for adult patients with chronic­ or accelerated­phase CML who are resistant to TKIs and other therapies; however, the underlying mechanisms remain unclear. In the present study, HHT treatment demonstrated induction of apoptosis in imatinib­resistant K562G cells by using MTS assay and western blotting, and BCR­ABL protein was reduced. CHX chase assay revealed that HHT induced degradation of the BCR­ABL protein, which could be reversed by autophagy lysosome inhibitors Baf­A1 and CQ. Next, HHT treatment confirmed the induction of autophagy in K562G cells, and silencing the key autophagic proteins ATG5 and Beclin­1 inhibited the degradation of the BCR­ABL protein and cytotoxicity. In addition, autophagic receptor p62/SQSTM1(p62) participated during the autophagic degradation of BCR­ABL induced by HHT, and this was confirmed by co­immunoprecipitation, in which HHT enhanced the ubiquitination of the BCR­ABL protein and promoted its binding to p62. In conclusion, HHT induced p62­mediated autophagy in imatinib­resistant CML K562G cells, thus promoting autophagic degradation of the BCR­ABL protein and providing a novel strategy for the treatment of TKI­resistant CML.

Synthesis, Biological Activities and Docking Studies of Novel 4-(Arylaminomethyl)benzamide Derivatives as Potential Tyrosine Kinase Inhibitors.

A number of new compounds containing the 4-(aminomethyl)benzamide fragment as a linker were designed and synthesized, and their biological activities were evaluated as potential anticancer agents. The cytotoxicity activity of the designed compounds was studied in two hematological and five solid cell lines in comparison with the reference drugs. Targeted structures against eight receptor tyrosine kinases including EGFR, HER-2, HER-4, IGF1R, InsR, KDR, PDGFRa, and PDGFRb were investigated. The majority of the compounds showed a potent inhibitory activity against the tested kinases. The analogues 11 and 13 with the (trifluoromethyl)benzene ring in the amide or amine moiety of the molecule were proven to be highly potent against EGFR, with 91% and 92% inhibition at 10 nM, respectively. The docking of synthesized target compounds for nine protein kinases contained in the Protein Data Bank (PDB) database was carried out. The molecular modeling results for analogue 10 showed that the use of the 4-(aminomethyl)benzamide as a flexible linker leads to a favorable overall geometry of the molecule, which allows one to bypass the bulk isoleucine residue and provides the necessary binding to the active center of the T315I-mutant Abl (PDB: 3QRJ).

Discovery of novel Bcr-AblT315I inhibitors with flexible linker. Part 1: Confirmation optimization of phenyl-1H-indazol-3-amine as hinge binding moiety.

As a continuation to our research, a series of novel Bcr-Abl inhibitors incorporated with 6-phenyl-1H-indazol-3-amine as hinge binding moiety (HBM) were developed based on confirmation analysis. Biological results indicated that these compounds exhibited an enhanced inhibition against Bcr-AblWT and Bcr-AblT315I in kinases assays, along with improved anti-proliferative activities in K562 cell assays. In particular, compound Y9 displayed comparable potency with that of imatinib. It potently inhibited Bcr-AblWT and Bcr-AblT315I kinases with IC50 of 0.043 µM and 0.17 µM, respectively. Furthermore, compound Y9 inhibited the proliferation of K562 and K562R cells with IC50 of 1.65 µM and 5.42 µM, respectively. Therefore, 6-phenyl-1H-indazol-3amine as HBM, combined with flexible linker, is a successful strategy contribute to research on T315I mutant resistance, and compound Y9 could be served as a starting point for further optimization.

Optimal Time Points for BCR-ABL1 Tyrosine Kinase Domain Mutation Analysis on the Basis of European LeukemiaNet Recommendations in Chronic Myeloid Leukemia.

BACKGROUND: In this study we aimed to evaluate appropriate time points for mutation analysis of chronic myeloid leukemia. PATIENTS AND METHODS: In total, 961 blood samples obtained from 605 chronic-phase chronic myeloid leukemia patients treated with imatinib were subjected to Sanger sequencing to detect BCR-ABL1 mutations. Mutation frequencies at landmark time points (3, 6, and 12 months) were assessed with 16 landmark responses defined by European LeukemiaNet and 2 additional responses, including a complete hematologic response (CHR) at 3 months and a complete cytogenetic response (CCyR) at 12 months. RESULTS: After 12 months of imatinib treatment of 605 patients, 28 (4.6%) patients harbored 33 mutations, including 23 (69.7%) highly resistant T315I and P-loop mutations. Sequencing data from 650 samples were compared with cytogenetic responses. The mutation frequencies in optimal, warning, and failure groups were 0.5% (2/430), 1.8% (2/110), and 19.1% (21/110), respectively. The molecular response was assessed using 956 samples, and the mutation frequencies were 0.7% (3/425), 3.4% (12/358), and 7.6% (14/173) for the optimal, warning, and failure groups, respectively. For the 2 additional responses, the mutation frequencies in patients with CHR at 3 months and with CCyR at 12 months were 0% (0/160) and 1.7% (4/242), respectively. Overall, mutations were frequently detected at 3-month cytogenetic failure (25.0%), 12-month cytogenetic failure (23.2%), and 6-month cytogenetic failure (10.5%) using response-mutation association analysis. CONCLUSION: Irrespective of mutation frequency, failure of achievement of a cytogenetic response should be conducted with appropriate mutation analysis.

Molecular dynamics investigation on the Asciminib resistance mechanism of I502L and V468F mutations in BCR-ABL.

Asciminib, a highly selective non-ATP competitive inhibitor of BCR-ABL, has demonstrated to be a promising drug for patients with chronic myeloid leukemia. It is a pity that two resistant mutations (I502L and V468F) have been found during the clinical trial, which is a challenge for the curative effect of Asciminib. In this study, molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GB/SA) calculations were performed to investigate the molecular mechanism of Asciminib resistance induced by the two mutants. The obtained results indicate that the mutations have adversely influence on the binding of Asciminib to BCR-ABL, as the nonpolar contributions decline in the two mutants. In addition, I502L mutation causes α-helix I' (αI') to shift away from the helical bundle composed of αE, αF, and αH, making the distance between αI' and Asciminib increased. For V468F mutant, the side chain of Phe468 occupies the bottom of the myristoyl pocket (MP), which drives Asciminib to shift toward the outside of MP. Our results provide the molecular insights of Asciminib resistance mechanism in BCR-ABL mutants, which may help the design of novel inhibitors.

Allosteric Modulator Discovery: From Serendipity to Structure-Based Design.

Allosteric modulators bound to structurally diverse allosteric sites can achieve better pharmacological advantages than orthosteric ligands. The discovery of allosteric modulators, however, has been traditionally serendipitous, owing to the complex nature of allosteric modulation. Recent advances in the understanding of allosteric regulatory mechanisms and remarkable availability of structural data of allosteric proteins and modulators, as well as their complexes, have greatly contributed to the development of various computational approaches to guide the structure-based discovery of allosteric modulators. This Perspective first outlines the evolution of the allosteric concept and describes the advantages and hurdles facing allosteric modulator discovery. The current available computational approaches, together with experimental approaches aiming to highlight allosteric studies, are then highlighted, emphasizing successful examples with their combined applications. We aimed to increase the awareness of the feasibility of the structure-based discovery of allosteric modulators using an integrated computational and experimental paradigm.

Compound mutations involving T315I and P-loop mutations are the major components of multiple mutations detected in tyrosine kinase inhibitor resistant chronic myeloid leukemia.

To analyze the pattern of multiple mutations detected by Sanger sequencing (SS), we performed subcloning sequencing using 218 samples from 45 patients with tyrosine kinase inhibitor resistant chronic myeloid leukemia. At the first time of multiple mutation detection by SS (baseline), a total of 19 major mutations from 45 samples were detected; these mutations were found in the following order: T315I (68.9%), E255 K (33.3%), Y253H (13.3%), G250E (13.3%), and F317 L (11.1%). Subcloning sequencing of 900 baseline colonies identified 556 different mutant types, and 791 among the 900 were colonies with major mutations (87.9%). The mutations were found in the following order: T315I (36.4%), E255 K (16.2%), Y253H (7.0%), G250E (6.7%), M351 T (6.6%), and E255 V (5.3%). In subcloning sequencing with 4357 colonies of 218 serial samples, 2506 colonies (57.5%) had compound mutations, among which 2238 colonies (89.3%) had at least one major mutation. The median number of mutations in compound mutant colonies was 2 (range, 2-7), and most were double (52.9%) or triple (28.7%) mutations. Additionally, some mutations in allosteric binding sites were detected as low level mutation in 13 patients. With the available retrospective samples before baseline, subcloning sequencing identified low-level mutations of various frequencies (median, 10%) to be major mutations in 20 patients. Thus, compound mutations involving T315I and P-loop mutations were the major components of multiple mutations, and some low-level mutations with potential clinical significance were detected by subcloning sequencing. Hence, more sensitive sequencing assays are needed in patients with multiple mutations.

Interaction of Abl Tyrosine Kinases with SOCS3 Impairs Its Suppressor Function in Tumorigenesis.

Suppressor of cytokine signaling 3 (SOCS3) is involved in Bcr-Abl-induced tumorigenesis. However, how SOCS3 interacts with Bcr-Abl and is regulated by Abl kinases remains largely unknown. Since c-Abl plays a critical role in tumorigenesis, we asked whether SOCS3 is regulated by c-Abl-dependent phosphorylation. Here, we found that SOCS3 interacted with all three Abl kinases (Bcr-Abl, v-Abl, and c-Abl), and SH1 domain of the Abl kinases was critically required for such interaction. Furthermore, the SH2 domain of SOCS3 was sufficient to pull down the SH1 domain but not the full length of Bcr-Abl. Importantly, SOCS3 was highly tyrosine phosphorylated by c-Abl, leading to impairment of its ability to suppress JAK8+72 activity. In addition, disrupting the tyrosine phosphorylation of SOCS3 promoted apoptosis of c-Abl-expressing cells and impeded xenograft growth of these tumor cells in nude mice. The results demonstrate that SOCS3 is highly tyrosine phosphorylated by c-Abl and that tyrosine phosphorylation of SOCS3 is required for the survival and tumorigenesis of certain cells. Our findings provide novel insights into complicated mechanisms underlying the oncogenic function of Abl kinases.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.