Role Of Protein-tyrosine Phosphatases In Improving Response To Second-generation TKI In CML Patients

Tyrosine kinase inhibitors for treating CML

Chronic myeloid leukemia (CML) occurs due to reciprocal chromosomal translocation t(9;22). The condition most commonly results in combination of the genes ABL1 (Abelson Murine Leukemia Viral Oncogene Homolog 1) and BCR (Breakpoint Cluster Region). This type of cancer affects the bone marrow and the blood cells and usually occurs at middle age. The abnormal fusion gene has been found responsible for the generation of a protein (usually p185 or p210) (1).

Owing to the fact that ABL1 gene comprises of a domain that has the capability of adding phosphate groups to residues of tyrosine, the fusion product BCE-ABL1 is also referred to as a tyrosine kinase. This oncogenic kinase facilitates the initiation of mitogenic signal transduction ways, subsequently leading to unrestrained propagation of myeloid cells and the progress of chronic myeloid leukemia.

Tyrosine kinase inhibitors (TKIs) that are directed towards BCR-ABL1 have proved as an effective treatment for CML patients. Improvements were found in the survival and remission rates, upon introduction of the first targeted therapy with imatinib. On the other hand, nilotinib and dasatinib that are second generation BCR-ABL1 TKIs that have been found to be stronger, thereby assisting several patients to attain a continued deep molecular response. The reason why the authors selected this topic can be accredited to the fact that despite huge improvement in treatment with the use of second-generation BCR-ABL1, a huge time is typically required for attaining molecular response. Thus, the primary aim of the researchers was to explore the role of Protein-tyrosine phosphatases (PTPs), in relation to second-generation TKI.

In the present study, the researchers examined 38 PTPs, collected from a cohort that comprised of 66 lately identified CML patients, who were treated with nilotinib (TIGER study). The research methodology encompassed the exploration of the effect of the PTP expression levels, prior to the envisioned beginning of the treatment. The effect was determined on the response, which in turn was reflected by the BCR-ABL1IS, 9 months following the treatment. An important aspect of the methodology involved selection of the PTPs, and subsequent analysis of their functional relevance on cell line models. This was concomitant with recognition of PTPRC and PTPRG as modulators of nilotinib intrinsic sensitivity, in relation to BCR-ABL1 expressing cells (1). Some of the key findings of the research were as follows:

1. Expression of PTP mRNA at the beginning of treatment is allied with molecular response in relation to nilotinib- Upon assessing the correlation between the mmRNA PTP levels with treatment response, with the help of univariate logistic regression, it was found that an equivalent number of patients with CML reached MR (BCR-ABL1IS ≤ 0.01 %; n = 30) and/or did not reach MR (BCR-ABL1IS > 0.01 %; n = 36). Increased mRNA levels for PTPRC (CD45) and PTPRG (RPTPγ), PTPRA (RPTPα), PTPN13 (FAP1, PTP-BAS), and PTPRM (RPTPµ) were associated with a noteworthy MR probability, following 9 months of treatment. Nonetheless, the results failed to demonstrate a significant association between PTPN2 (TC-PTP), PTPN6 (SHP-1), PTPN1 (PTP1B), and PTPN22 (Lyp). An assessment of the patient subgroups, exclusive of pre-treatment was also done, followed by categorization of two subgroups made up of 35/66 nd 54/66 patients (1).
2. PTPRG and PTPRC, and not PTPN6 control nilotinib response, in relation to cell lines- The succeeding analyses focused on PTPRC aand PTPRG, of the five different PTPs that were investigated earlier. Owing to the fact that PTPRG had shown negative regulation of BCR-ABL1 arbitrated transformation, the researchers aimed to focus their investigation on the same. On manipulating the expression level of the three PTP in the selected CML cell line, the levels of PTPRG were found to be considerably low in the primary cells and K562 cells. Overexpression of the wild type PTPRG and/or catalytically inactive PTPRG-C1060S (CS) mutant in K562 cell was followed by an analysis of the impacts on responsiveness of TKI. It was found that overexpression of PTPRG-WT resulted in an enhanced consequence of nilotinib and imatinib. This was confirmed by low IC50, in contrast to the lack of significant difference between effects of TKI on PTPRG-CS mutant-expressing cell, in comparison to controls. Increased expression of PTRPC were found on the CML primary cells. PTPRC knockout clone analysis revealed that an enhanced response was found, in relation to imatinib and nilotinib, which in turn was indicated by a decreased IC50. Inconsistent changes were observed on the two varied shRNAs, in accordance to TKI response. However, increased sensitivity was noted for nilotinib and imatinib for one particular shRNA. The manipulations were also not associated with changes in any three TKIs that were tested (1).
3. PTPRG attenuates and PTPRC encourages BCRABL1 dependent cell transformation- The researchers found that PTPRG-WT increased expression in the K562 cells were responsible for moderately attenuating formation of colonies, in contrast to the less significant impact of PTPRG-CS expression. Furthermore, the CRISPR/Cas9 facilitated PTPRC knockout was related with a concentrated colony development of engineered cells, present in methylcellulose. Exogenous PTPRC also heightened the extent of colony formation. This provided sufficient evidence for the attenuation of BCR-ABL1 dependent cell transformation by PTPRG (1).
4. Nilotinib treatment, PTPRG overexpression, and PTPRC deficiency influence BCR-ABL1 signal transduction in similar direction- PTPRG-WT and not PTPRG-CS overexpression created an inhibitory impact on autophosphorylation of BCR-ABL1, which in turn was evidence in serum absence and SFK Lyn phosphorylation. PTPRG-WT was found to moderately reduce ERK 1/2 phosphorylation, in the presence of serum. However, no significant impacts were observed for STAT5 activation. Furthermore, the loss of PTPRC caused a marked reduction of SFK activity, comprehended conspicuously upon examination, in absence of serum. This led to the conclusion that PTPRC plays a potential role in modulating the BCR-ABL1 signaling pathways, both positively and negatively, by differentially creating an impact on the output of specific signaling (1).

The research was the first of its kind that was able to provide a detailed and comprehensive account of the potential relation between expression of PTP and the response of CML cell lines and patients to second-generation TKI nilotinib. The researchers were successful in the identification of a range of PTPs whose expression was linked with more competent reaction to TKI, counting in PTPRC and PTPRG. The use of genetically modified cells, PTPRC and PTPRG were found to create a causal impact on the sensitivity of nilotinib at the cellular level. Overexpression of PTPRG and knockout of PTPRC with the use of CRISPR/Cas9 technology improved the nilotinib response. Significant correlation with the MR4 probabilities were also established by the expression levels of PTPRA, PTPN13, and PTPRM, 9 months following the event, in the cohort that comprised of 66 patients. This in turn provided further indication for the fact that there is a need to determine and explore the significance of PTP gene expression for responses related to TKI in CML, and possible purposes of PTPs at cellular level. Hence, it could be concluded that PTPs play a vital role in altering response to TKI in CML cells, in relation to second-generation nilotinib TKI. PTPRC and PTPRG impact TKI sensitivity in conflicting manner, in accordance with their influence on BCR-ABL1 cell transformation and signal transduction.

Research methodology

Background- Scientific evidences have established the importance of autophagy in IL-1 cytokine secretion that controls inflammatory disease development. Chloroquine (CHQ), the autophagy/lysome inhibitor and antimalarial drug has also been found to act as a probable trigger for drug aggravated psoriasis. In this particular condition the Th17 cells withstand a tenacious inflammation (2).

Aim- To determine the consequence of CHQ on dendritic cells (MoDC) and human monocyte-derived Langerhans-like cells (MoLC), in relation to IL-1b.

Methods- Several steps were involved in the research namely, (i) In vitro synthesis of DC and LC-like cells from human monocytes (MoLC and MoDC), (i) in vitro stimulation of MoDC and MoLc, (iii) autophagy blocking experiments, (iv) cell viability determination, (v) co-culture of stimulated CD4+ T cells and MoLC cells, (vi) Intracellular cytokine staining, (vii) ELISA, RNA isolation and quantitative RT-PCR, (viii) Immunofluorescence and LC3A punctae quantification, and (ix) statistical analysis.

Results- The incidence of CHQ condensed IL-12p70 release in both subcategories, but unexpectedly augmented IL-6 assembly in MoDC and IL-23 in MoLC. Prominently, CHQ-treated MoLC endorsed IL-17A emission by CD4+ T cells, followed by an increase in the RORC mRNA levels. In contrast, IFN-g release considerably decreased. IL-12 family cytokines dysregulation in MoDC and MoLC happened at transcriptional levels. Comparable influecnes were acquired with late autophagy inhibitors. Nonetheless, PI3K inhibitor 3-methyladenine was unsuccessful in increasing IL-23 secretion.

Conclusion- CHQ moderates release of cytokine in a p38-dependent manner, thereby signifying an indispensable part of dendritic cells and Langerhans cells in CHQ-provoked psoriasis, conceivably by indorsing Th17 immunity.

Autophagy had been found imperative in generation of immune responses against intracellular pathogens. The research is based on prior evidences that have also confirmed the role of autophagy in the regulation of discrete immunologic processes that are indispensable for the generation of powerful adaptive and innate immune responses. There exists a multifaceted interaction between cytokines and autophagy. Regardless of the role of Th2-related IL-13 and IL-4 in inhibiting autophagy, IL-1 activates the process. Furthermore, autophagosomes are also responsible for moderating the discharge of IL-1b, which in turn are related to IL-23 secretion. There is little evidence to explain the mechanism of autophagy in mediating immune response regulation in vivo. Nonetheless, Th1 and Th17 cells have been identified as essential intermediaries of inflammatory bowel disease pathology. In vivo Th17 cell differentiation is typically controlled by IL-6 and TGF-β, which in turn are under the influence of IL-1 cytokine release (2).

The fact that autophagy probably plays a role in maintaining the balance of Th1/Th17 in Crohn’s disease and associated inflammatory illnesses, formed the foundation for this research. Furthermore, though psoriasis has been documented as a germ-free inflammatory skin disease, there are strong evidences for the influence of Th17 on the disease pathogenesis that is marked by an increase in the levels of IL-1 cytokines in hyperproliferative tissue. Therefore, the research was based on the fact that a dysregulated IL-1 cytokine release provides an indication for a modified autophagic fluctuation in psoriasis phenotype related diseases.

After statistical analysis the following results were obtained:

1. CHQ inhibits IL-12p70 release and reduces Th1-priming capacity of activated MoLC- The research was based on previous successful use of CHQ in therapies for the treatment of inflammatory diseases that resulted in a decrease in the release of IFN-g, mediated by CD4+ and CD8+ T cells and secretion of proinflammatory cytokines. Proinflammatory cytokines rh-TNF-α and rh-IL-1β were used in combination with LPS for inducing maturation, with the aim of exploring the role of CHQ treatment on Th1 cytokine IL-12p70 release by activated MoLC or MoDC. Owing to the decrease in responsiveness of LC, in relation to bacterial Ags, the researchers found that mature MoLC demonstrated a reduced effectiveness in triggering release of IL-12p70, when compared to MoDC. On assessing Thi-priming capacity with the help of naïve CD4+ T cells, greater levels of IFN-g were found in the cell culture supernatants, which when restimulated with the help of ionomycin and PMA, established an upregulation in type 1 T cell response. On the other hand, CHQ presence during MoLC activation totally repressed IFN-g production that was T cell–induced. Moreover, RORC mRNA levels were somewhat upregulated on pretreating MoLC with CHQ (2).
2. CHQ augments IL-1–induced IL-23 release in MoLC and consequently upsurges IL-17A secretion by primed CD4+ T cells- The research was conducted with the aim of characterizing the consequence of CHQ on the expansion and progress of Th17 cells. This made the researchers add Th17 cytokines (rh-IL-1b, rh-IL-6, and rh-TGF-b1) during naive CD4+ T cells and activated MoLC coculture. Results revealed an increase in the levels of IL-17A, in comparison to T cells cultivation, under circumstances that were marked by an absence of Th17- priming cytokines. Upon analysis the priming capacity of MoLC that were pretreated with CHQ, an increase in IL-17A secretion by corresponding CD4+ T cells was observed, upon restimulation. This was in relation to conditions that were marked by presence of CHQ during MoLC activation, thereby indorsing a likely modification of Th cell reactions from Th1 (IFN-g) to Th17. The findings also revealed the role of rh-IL-1β in successive stimulation of untreated MoLC. This is in clear contrast to the fact that CHQ presence during IL-1β stimulation significantly resulted in an elevation in the release of IL23 by MoLC, in place of MoDC that was considerably decreased or unaffected (2).
3. MoDC increases IL-6 secretion in presence of CHQ- In addition to IL-23, which was had already been established imperative for the preservation of Th17 pathogenic phenotype, IL-6 was found to induce differentiation of Th17 in vivo, concomitant with IL-1 and TGF-β cytokines. Upon conducting an intracellular FACS analysis an association was confirmed between activated MoDC and IL-6+ population, which in turn demonstrated an increase on subjecting them to CHQ treatment. Additionally, p40 subunit costaining demonstrated a reduction in p40+ cells, in presence of CHQ, thus approving the decreased IL-12p70 levels in all subsets (2).
4. CHQ differentially regulates gene expression of IL-12 family cytokines- Upon assessing the differential regulation IL-12 cytokine family release, no effects on the p35 mRNA levels were obtained, in relation to stimulation with rh-IL-1β. Hence, it provided evidence for the fact that stimulation with only rh-IL1β does not induce release of IL-12p70 in both subsets. The upregulation of p40 and p19 mRNA levels in stimulated MoLC established the heightened release of IL-23 in presence of CHQ. Likewise, treatment with 3-MA failed to improve IL-23 secretion, though both subsets reasonably upregulated p19 mRNA.
5. Inhibition of IL-1R rescinds IL-23 release in stimulated MoLC, while TGF-bR1 signaling seems dispensable- It was found that secretion of IL-23 was largely reliant on the initiation of IL-1R signaling. The researchers had used hydrocinnamoyl-L-valyl pyrrolidine (IL-1Ra) that acted as a precise inhibitor of MyD88/IL-1R1 interaction. It was found that there was a marked reduction in the secretion of IL-23, induced by either rh-IL-1β or rh-IL-1α, in presence of snowballing IL-1Rα concentrations in CHQ-treated MoLC. Furthermore, no significant alterations were found in rh-IL1β associated release of IL-23 in MoLC that were treated with CHQ.
6. CHQ elevates IL-23 and IL-6 secretion in the presence of TLR2/1 ligand- MoDC was found to be responsible for an increased upregulation of IL-6 in presence of CHQ. In contrast, initiation of TLR2/1 in CHQ presence resulted in an upsurge in secretion of IL-23 in MoLC (2).
7. LC3-II, p62/SQSTM1, and TRAF6 expression is increased in the presence of CHQ- Results obtained from immunofluorescence analysis discovered a reduction in the amounts of endogenous LC3A-positive vesicles, upon treatment with PI3K inhibitor 3-methyladenine (3-MA).

The differential control of cytokines in presence of CHQ designated a crucial stimulatory function of dermal DC and LC in psoriasis that was drug-provoked, by upholding a pathological IL23/ Th17/ IL-6 axis in inflammatory conditions, marked by raised IL-1 cytokine levels.

References

1. Drube J, Ernst T, Pfirrmann M, Albert BV, Drube S, Reich D, Kresinsky A, Halfter K, Sorio C, Fabisch C, Hochhaus A. PTPRG and PTPRC modulate nilotinib response in chronic myeloid leukemia cells. Oncotarget. 2018 Feb 6;9(10):9442.
2. Said A, Bock S, Lajqi T, Müller G, Weindl G. Chloroquine promotes IL-17 production by CD4+ T cells via p38-dependent IL-23 release by monocyte-derived Langerhans-like cells. The Journal of Immunology. 2014 Nov 10:1303276.

Order an Essay Now & Get These Features For Free:

Turnitin Report

Formatting

Title Page

Citation

Outline

Place an Order