Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

Parallel Chemoselective Profiling for Mapping Protein Structure.

Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques.

How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture.

Small molecule kinase inhibitors that stabilize distinct ATP binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects generalize to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src's regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix αC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src's helix αC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Our study highlights that an ATP-competitive inhibitor's interactions with helix αC can have a major influence on the global conformation of some tyrosine kinases.

Chemoproteomic Method for Profiling Inhibitor-Bound Kinase Complexes.

Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein's functions affects the state of the cell is not well-understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. Here, we describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src's cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src's interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src's localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets.

Targeting Dynamic ATP-Binding Site Features Allows Discrimination between Highly Homologous Protein Kinases.

ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.

A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Allosteric Modulation of Src Family Kinases with ATP-Competitive Inhibitors.

The Src family kinases (SFKs) are an important family of tyrosine kinases that are allosterically regulated by their SH2 and SH3 domains. Engagement of SFK SH2 and SH3 domains with their intramolecular ligands leads to reduced kinase activity by stabilizing an inactive ATP-binding site conformation. Disruption of these intramolecular interactions stabilizes a more active ATP-binding site conformation and restores SFK activity. Interestingly, this allosteric relationship is bidirectional in that ATP-competitive ligands that stabilize distinct active site conformations can divergently modulate the abilities of the regulatory SH2 and SH3 domains to participate in intermolecular interactions. Here, we describe a series of assays that profile the bidirectional relationship between the ATP-binding sites and regulatory domains of SFKs. These methods can be used to discover ATP-competitive inhibitors that are selective for distinct ATP-binding site conformations of SFKs and for characterizing the effects that ATP-competitive inhibitors of SFKs have on domains that are distal to their site of interaction.

Cytogenetic features of human trophoblast cell lines SWAN-71 and 3A-subE.

Immortalization of primary cells with telomerase is thought to maintain normal phenotypic properties and avoid chromosomal abnormalities and other cancer-associated changes that occur following simian virus 40 tumor antigen (SV40 Tag) induced immortalization. However, we report that the human telomerase reverse transcriptase (hTERT)-immortalized SWAN-71 trophoblast cell line has a near pentaploid 103∼119,XXXX[cp20] karyotype. Additionally, DNA typing analysis indicated that SWAN-71 cells have acquired microsatellite instability. In comparison, the post-crisis SV40-transformed trophoblast cell line 3A-subE was hypertriploid 69∼81,XX[cp20]. Both cell lines contained multiple specific clonal rearrangements. These findings emphasize the need to monitor for genetic instability in hTERT-immortalized cells.

Molecular Diagnosis of Myotonic Dystrophy.

Myotonic dystrophy types 1 (DM1) and 2 (DM2) are autosomal dominant, microsatellite repeat expansion disorders that affect muscle function. Myotonic dystrophy type 1 is caused by CTG repeat expansion in the 3' UTR region of the DMPK gene. Patients with DM2 have expansion of CCTG repeats in intron 1 of the CNBP gene. In this unit, we review and discuss the clinical phenotypes, genetic mutations causing the diseases, and the molecular diagnostic approaches and tools that are used to determine repeat sizes in DM1/2. In summary, the goal of this chapter is to provide the reader with a basic understanding of the clinical, genetic and diagnostic aspects of these disorders. © 2016 by John Wiley & Sons, Inc.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene.

BACKGROUND: Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. OBJECTIVE: Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. METHODS: Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. RESULTS: Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. CONCLUSIONS: XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

Therapy-related myeloid neoplasm with bone marrow involvement, myelosarcoma, and a t(8;16)(p11.2;p13.3)-a case report.

Therapy-related leukemia is a well-documented complication of conventional therapy for cancer. Therapy-related acute myeloid leukemia (t-AML) is grouped along with therapy-related myelodysplastic syndrome (t-MDS) and therapy-related myelodysplastic syndrome/myeloproliferative neoplasms (t-MDS/MPNs) as therapy-related myeloid neoplasms (t-MNs) by the 2008 World Health Organization classification system. Therapy-related myeloid neoplasms differ clinically from their de novo counterparts in terms of response to therapy, aggressiveness of disease, and associated poor prognosis. The occurrence of extramedullary myeloid sarcomas with bone marrow involvement has been shown to be a poor prognostic indicator for patients with t-MN. The karyotype of leukemic blasts has also been reported to have a significant impact in t-MN and may predict survival and outcomes in patients. The t(8;16)(p11.2;p13.3) is a rare, balanced translocation that is frequently associated with the M4/M5 subtype of de novo acute myeloid leukemia. It has also been reported in patients with t-MN, typically in those with poor outcomes. Here we report a case of t-MN with myeloid sarcoma and bone marrow involvement in an adult patient with a karyotype of 47,XY,t(8;16)(p11.2;p13.3),+21 after rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemotherapy for follicular lymphoma.

Chronic myelogenous leukemia stem and progenitor cells demonstrate chromosomal instability related to repeated breakage-fusion-bridge cycles mediated by increased nonhomologous end joining.

Chromosomal aberrations are an important consequence of genotoxic exposure and contribute to pathogenesis and progression of several malignancies. We investigated the susceptibility to chromosomal aberrations in chronic myelogenous leukemia (CML) progenitors after exposure to ionizing radiation. In normal progenitors, ionizing radiation induced both stable and unstable chromosomal lesions, but only stable aberrations persisted after multiple divisions. In contrast, radiation of chronic phase CML progenitors resulted in enhanced generation of unstable lesions that persisted after multiple divisions. CML progenitors demonstrated active cell cycle checkpoints and increased nonhomologous end joining DNA repair, suggesting that persistence of unstable aberrations was the result of continued generation of these lesions. CML progenitors demonstrated enhanced susceptibility to repeated cycles of chromosome damage, repair, and damage through a breakage-fusion-bridge mechanism. Perpetuation of breakage-fusion-bridge cycles in CML progenitors was mediated by classic nonhomologous end joining repair. These studies reveal a previously unrecognized mechanism of chromosomal instability in leukemia progenitors because of continued generation of unstable chromosomal lesions through repeated cycles of breakage and repair of such lesions.

Altered hematopoietic cell gene expression precedes development of therapy-related myelodysplasia/acute myeloid leukemia and identifies patients at risk.

Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a major complication of cancer treatment. We compared gene expression in CD34+ cells from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for lymphoma with controls who did not develop t-MDS/AML. We observed altered gene expression related to mitochondrial function, metabolism, and hematopoietic regulation in pre-aHCT samples from patients who subsequently developed t-MDS/AML. Progression to overt t-MDS/AML was associated with additional alterations in cell-cycle regulatory genes. An optimal 38-gene PBSC classifier accurately distinguished patients who did or did not develop t-MDS/AML in an independent group of patients. We conclude that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and accurately identify patients at risk for this complication.

Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment.

Imatinib mesylate treatment markedly reduces the burden of leukemia cells in chronic myelogenous leukemia (CML) patients. However, patients remain at risk for relapse on discontinuing treatment. We have previously shown that residual BCR-ABL(+) progenitors can be detected in CML patients within the first 2 years of imatinib treatment. However, reduced rates of relapse and continued decline of BCR-ABL levels with prolonged treatment, together with the ability of selected patients to maintain remission after discontinuing treatment, led us to investigate whether prolonged imatinib exposure resulted in reduction or elimination of BCR-ABL(+) stem cells. We evaluated BCR-ABL expression in CD34(+)CD38(+) (38(+)) committed progenitors and CD34(+)CD38(-) (38(-)) stem/primitive progenitor cells in samples from CML patients on imatinib treatment for at least 4 years with cytogenetic and molecular response. High levels of BCR-ABL expression were maintained over time in the 38(-) stem cell fraction. The absolute frequency of BCR-ABL(+) cells as determined by limiting dilution analysis was consistently higher in 38(-) compared with 38(+) cells. Transplantation into NOD/SCID-IL2Rγ-chain knockout mice demonstrated that BCR-ABL(+) cells had long-term in vivo repopulating capacity. These results directly demonstrate that BCR-ABL(+) stem cells persist in CML patients despite prolonged treatment with imatinib, and support ongoing efforts to target this population.

Accelerated telomere shortening precedes development of therapy-related myelodysplasia or acute myelogenous leukemia after autologous transplantation for lymphoma.

PURPOSE: Therapy-related myelodysplasia or acute myelogenous leukemia (t-MDS/AML) is a lethal complication of autologous hematopoietic stem-cell transplantation (aHCT) for Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Here, we investigated the hypothesis that accelerated telomere shortening after aHCT could contribute to the development of t-MDS/AML. PATIENTS AND METHODS: A prospective longitudinal cohort was constructed to investigate the sequence of cellular and molecular abnormalities leading to development of t-MDS/AML after aHCT for HL/NHL. This cohort formed the sampling frame for a nested case-control study to compare changes in telomere length in serial blood samples from patients who developed t-MDS/AML with matched controls who did not develop t-MDS/AML. RESULTS: An initial increase in telomere length at day 100 after aHCT was followed by an accelerated telomere shortening in t-MDS/AML patients when compared with controls. These telomere alterations preceded the onset of t-MDS and were independent of other known risk factors associated with development of t-MDS/AML on multivariate analysis. Additionally, we observed reduced generation of committed progenitors in patients who developed t-MDS/AML, indicating that these telomere alterations were associated with reduced regenerative capacity of hematopoietic stem cells. CONCLUSION: The development of t-MDS/AML after aHCT is associated with and preceded by markedly altered telomere dynamics in hematopoietic cells. Accelerated telomere loss in patients developing t-MDS/AML may reflect increased clonal proliferation and/or altered telomere regulation in premalignant cells. Genetic instability associated with shortened telomeres may contribute to leukemic transformation in t-MDS/AML.