Tyrosine Phosphorylation Of CrkL Research Articles

Safety and Efficacy of HQP1351, a 3rd Generation Oral BCR-ABL Inhibitor in Patients with Tyrosine Kinase Inhibitor—Resistant Chronic Myelogenous Leukemia: Preliminary Results of Phase I Study

Background: HQP1351, a novel, potent 3rd generation TKI, was designed to treat patients with chronic myeloid leukemia (CML) resistant to current TKI-therapies including those with T315I mutation.Aims: A single-agent, open-label dose escalation and dose expansion Phase I study to assess the safety, preliminary efficacy, pharmacokinetic (PK) and pharmacodynamic (PD) properties of orally administered HQP1351 in the TKI-resistant patients with chronic phase (CP) or accelerated phase (AP) CML.Methods: HQP1351 was administered once every other day (QOD) in 28-days cycles at 11 dose cohorts ranging from 1mg to 60 mg. The eligible patients received treatments until disease progression or intolerable toxicities. The primary efficacy endpoint in the CML AP and CP patients, was complete hematological response (CHR) and major cytogenetic response (MCyR) respectively, MCyR includes partial cytogenetic response (PCyR) and complete cytogenetic response (CCyR).Blood samples were collected at various time points on Day 1-2 and Day 27-28 during cycle 1 for PK analyses. BCR-ABL inhibition was evaluated using tyrosine phosphorylation of CRKL and STAT5 in peripheral blood mononuclear cell (PBMCs) collected from the patients before and 4, 8, 24 and 48 hours post HQP1351 treatments on Day 1, 15 and 27 during cycle 1.Results:From November 2016 to July 2018, 70 patients (CP n=58 and AP n=12) enrolled received ≥1 cycle of HQP1351-treatment, only 1 patient withdrew from the study due to disease progression. Median age was 39 (range: 23-59) years. Median interval from CML diagnosis to starting HQP1351-treatment was 6.1 (1.1-14.7) years. Sixty-one (87%) patients received ≥2 prior lines of TKI-therapy. Fifty-three (76%) patients had BCR-ABL mutations and 45 (64%) had T315I mutation at baseline.With a median follow-up of 4.1 (1.0-21.2) cycles, HQP1351 was well-tolerated in all dose cohorts other than the 60mg cohort. In all patients, 67 (96%) patients experienced ≥1 treatment related adverse events (TRAEs) and 44 (63%) experienced TRAE(s) of grade 3-4 (Table 1). There was no patient withdrawal from the study because of TRAEs. Two out of 3 patients in the 60mg cohort experienced dose-limiting toxicity(DLT) and HQP1351 at 50mg QOD was considered as MTD.The anti-leukemic activities of HQP1351 were observed in this study. Sixty-five (93%) patients including 58 (100%) CP and 7 (58%) AP patients achieved CHR at the dose of 2mg to 60mg within 3 cycles. In 47 evaluable patients receiving HQP1351-treatment ≥3 cycles, 24 (51%) achieved MCyR including 21 (54%) CP and 3 (38%) AP patients at the dose of 12mg to 50mg, and 14 (30%) patients achieved CCyR including 12 (31%) CP and 2 (25%) AP patients. Total 6 (15%) CP patients achieved MMR. More than 65% of the patients achieved MCyR or MMR at the end of cycle 3. HQP1351 was highly active in patients with or without T315I mutation at baseline (Table 2).Following oral administration of HQP1351 at doses ranging from 1mg to 60 mg, the peak concentration was reached at 2-8 hrs. The elimination appeared to be linear with a mean terminal T1/2 of 15.3-36.5 hrs on Day 1, 18.8-42.5 hrs on Day 27, respectively. The ratios for AUC0-t and Cmax of HQP1351 on Day 27 versus Day 1 ranged from 1.03 to 2.12 and from 0.78 to 1.93, respectively. Thus, HQP1351 exhibited an approximately dose proportional increase in Cmax and AUC0-t following single or multiple oral administration dose ranging from 1 to 60 mg. PD study results demonstrated that reduction of CRKL phosphorylation was schedule and dose-dependent, ≥50% reduction was observed at doses ranging 12mg-60mg.Conclusions: The preliminary results of the phase 1 study showed that HQP1351, a novel 3rd-generation TKI, is safe and highly active in treatment of the TKI-resistant patients with CP-CML and AP-CML,with or without T315I mutation. DisclosuresChen:HealthQuest Pharma Inc.: Employment. Men:HealthQuest Pharma Inc.: Employment. Liu:HealthQuest Pharma Inc.: Employment. Sun:HealthQuest Pharma Inc.: Employment. JI:HealthQuest Pharma Inc.: Employment. Wang:HealthQuest Pharma Inc.: Employment. Hu:HealthQuest Pharma Inc.: Employment. Zou:HealthQuest Pharma Inc.: Employment. Yan:HealthQuest Pharma Inc.: Employment. Huang:Ascentage Pharma Group Inc.: Employment; HealthQuest Pharma Inc.: Employment. Yang:Ascentage Pharma Group Inc.: Employment. Zhai:Ascentage Pharma Group Inc.: Employment.

CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma.

Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients.

Glucocorticosteroid Enhances the Tyrosine Phosphorylation of CrkL in Antigen-stimulated Mast cells

Glucocorticosteroid Enhances the Tyrosine Phosphorylation of CrkL in Antigen-stimulated Mast cells

BCR/ABL P190 transgenic mice develop leukemia in the absence of Crkl.

The Bcr/Abl fusion protein directly causes chronic myelogenous leukemia and Philadelphia-chromosome positive acute lymphoblastic leukemia. Multiple independent studies have implicated Crkl, a small adapter protein, in transduction of oncogenic signals of Bcr/Abl and Crkl tyrosine-phosphorylation is used as a diagnostic tool for Philadelphia-positive leukemia. To evaluate the contribution of Crkl to this type of leukemia, we generated mutant mice that lack Crkl expression. We found that the overall survival of P190 BCR/ABL crkl-/- mice was comparable to that of genetically matched P190 BCR/ABL crkl +/+ mice. Both genotypes developed lymphoid lineage leukemia/lymphoma. Western blot analysis of -/- and +/+ lymphomas showed that the related Crk protein was tyrosine phosphorylated and could be found complexed with Bcr-Abl P190. These data indicate that possible therapeutic approaches that target Crkl may be complicated by the presence of pathways that compensate for lack of Crkl function.

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation.

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 x 10(9)/l. P210BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p210BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.

The tetramerization domain-independent Ras activation by BCR-ABL oncoprotein in hematopoietic cells.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that BCR-ABL activates Ras signaling pathways required for transformation. To elucidate the mechanisms whereby BCR-ABL induced transformation in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. We found that the Grb2 binding site-deleted BCR-ABL and the SH2 domain-deleted BCR-ABL, as well as the tetramerization domain-deleted BCR-ABL do not diminish the transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. The tetramerization domain-deleted BCR-ABL did not induce tyrosine phosphorylation of CrkL, SHP-2, Vav and the interactions of BCR-ABL and Shc. However, Ras is activated, Shc is tyrosine phosphorylated and binds to Grb2 in the tetramerization domain-deleted BCR-ABL expressing hematopoietic cells. These results suggest that the tetramerization domain-independent Ras activation is mediated by Shc proteins and induces the transformation of hematopoietic cells.

Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site.

BCR/ABL has a causal role in Philadelphia (Ph)-chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation.

Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.