Comprehensive analysis of the HCK gene in myeloid neoplasms: Insights into biological functions, prognosis, and response to antineoplastic agents.

Myeloid neoplasms result from molecular alterations in hematopoietic stem cells, with acute myeloid leukemia (AML) being one of the most aggressive and with a poor prognosis. Hematopoietic cell kinase (HCK) is a proto-oncogene that encodes a protein-tyrosine kinase of the Scr family, and it is highly expressed in AML. The present study investigated HCK expression in normal hematopoietic cells across myeloid differentiation stages and myeloid neoplasm patients. Within the AML cohort, we explored the impact of HCK expression on clinical outcomes and its correlation with clinical, genetic, and laboratory characteristics. Furthermore, we evaluated the association between HCK expression and the response to antineoplastic agents using ex vivo assay data from AML patients. HCK expression is higher in differentiated subpopulations of myeloid cells. High HCK expression was observed in patients with chronic myelomonocytic leukemia, chronic myeloid leukemia, and AML. In patients with AML, high levels of HCK negatively impacted overall and disease-free survival. High HCK expression was also associated with worse molecular risk groups and white blood cell count; however, it was not an independent prognostic factor. In functional genomic analyses, high HCK expression was associated with several biological and molecular processes relevant to leukemogenesis. HCK expression was also associated with sensitivity and resistance to several drugs currently used in the clinic. In conclusion, our analysis confirmed the differential expression of HCK in myeloid neoplasms and its potential association with unfavorable molecular risks in AML. We also provide new insights into HCK biological functions, prognosis, and response to antineoplastic agents.

The antitumor effects of WNT5A against hematological malignancies.

The bone marrow (BM) microenvironment (niche) is abnormally altered in acute myeloid leukemia (AML), leading to deficient secretion of proteins, soluble factors, and cytokines by mesenchymal stromal cells (MSC) that modifies the crosstalk between MSC and hematopoietic cells. We focused on a WNT gene/protein family member, WNT5A, which is downregulated in leukemia and correlated with disease progression and poor prognosis. We demonstrated that WNT5A protein upregulated the WNT non-canonical pathway only in leukemic cells, without modulating normal cell behavior. We also introduced a novel WNT5A-mimicking compound, Foxy-5. Our results showed reduction of crucial biological functions that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy, as well as G0/G1 cell cycle arrest. Additionally, Foxy-5 induced early-stage macrophage cell differentiation, a crucial process during leukemia development. At a molecular level, Foxy-5 led to the downregulation of two overexpressed leukemia pathways, PI3K and MAPK, which resulted in a disarrangement of actin polymerization with consequent impairment of CXCL12-induced chemotaxis. Notably, in a novel tri-dimensional bone marrow-mimicking model, Foxy-5 led to reduced leukemia cell growth and similar results were observed in a xenograft in vivo model. Overall, our findings highlight the pivotal role of WNT5A in leukemia and demonstrate that Foxy-5 acts as a specific antineoplastic agent in leukemia, counterbalancing several leukemic oncogenic processes related to the crosstalk in the bone marrow niche, and represents a promising therapeutic option for AML. WNT5A, a WNT gene/protein family member, is naturally secreted by mesenchymal stromal cells and contributes to the maintenance of the bone marrow microenvironment. WNT5A downregulation is correlated with disease progression and poor prognosis. The treatment with Foxy-5, a WNT5A mimetizing compound, counterbalanced several leukemogenic processes that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy and disruption of PI3K and MAPK signaling pathways.

Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues.

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.

Visible intestinal peristalsis and chronic diarrhea due to a rare lymphoproliferative disease.

We present the case of a 45-year-old man with watery diarrhea for 2 years, leading to marked weight loss (52 kg). On admission, the patient presented with pallor, dehydration and cachexia. Abdominal examination revealed increased bowel sounds, painful and visible intestinal peristalsis, suggesting intestinal obstruction. There was no response to a gluten-free diet and nutritional support. Finally, the patient developed pulmonary infection, septic shock and died 3 months after admission. The diagnosis of CD4+/CD8+ indolent T-cell lymphoma of the GI tract was made post-mortem.

WNT5A in tumor development and progression: A comprehensive review.

The investigation of tumor microenvironment (TME) is essential to better characterize the complex cellular crosstalk and to identify important immunological phenotypes and biomarkers. The niche is a crucial contributor to neoplasm initiation, maintenance and progression. Therefore, a deeper analysis of tumor surroundings could improve cancer diagnosis, prognosis and assertive treatment. Thus, the WNT family exerts a critical action in tumorigenesis of different types of neoplasms due to dysregulations in the TME. WNT5A, an evolutionary WNT member, is involved in several cellular and physiopathological processes, in addition to tissue homeostasis. The WNT5A protein exerts paradoxical effects while acting as both an oncogene or tumor suppressor by regulating several non-canonical signaling pathways, and consequently interfering in cell growth, cytoskeletal remodeling, migration and invasiveness. This review focuses on a thorough characterization of the role of WNT5A in neoplastic transformation and progression, which may help to understand the prognostic potentiality of WNT5A and its features as a therapeutic target in several cancers. Additionally, we herein summarized novel findings on the mechanisms by which WNT5A might favor tumorigenesis or suppression of cancer progression and discussed the recently developed treatment strategies using WNT5A as a protagonist.

Novel inhibitor of hematopoietic cell kinase as a potential therapeutic agent for acute myeloid leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine.

Hematopoietic Cell Kinase (HCK) Is a Player of the Crosstalk Between Hematopoietic Cells and Bone Marrow Niche Through CXCL12/CXCR4 Axis.

The crosstalk between hematopoietic stem/progenitor cells (HSC), both normal and leukemic, and their neighboring bone marrow (BM) microenvironment (niche) creates a reciprocal dependency, a master regulator of biological process, and chemotherapy resistance. In acute myeloid leukemia (AML), leukemic stem/progenitor cells (LSC) anchored in the protective BM microenvironment, reprogram and transform this niche into a leukemia-supporting and chemoprotective environment. One most important player involved in this crosstalk are CXCL12, produced by the BM mesenchymal stromal cells, and its receptor CXCR4, present onto HSC. The downstream molecular mechanisms involved in CXCL12/CXCR4 axis have many targets, including the Src family members of non-receptor tyrosine kinase (SFK). We herein study the role of one SFK member, the Hematopoietic Cell Kinase (HCK), in CXCL12/CXCR4 pathway and its contribution to the AML pathogenesis. We verified that the inhibition of HCK severely impaired CXCL12-induced migration of leukemic cell lines and CD34 positive cells from AML patients bone marrow, through a disruption of the activation of CXCL12/CXCR4/PI3K/AKT and CXCL12/CXCR4/MAPK/ERK signaling, and by a decreased cytoskeleton dynamic through a lower rate of actin polymerization. We provide new insights into the key role of HCK in conferring a migratory advantage to leukemic cells thought CXCL12/CXCR4 axis. HCK represents an important protein of the main pathway involved in the crosstalk between HSC, and their surrounding milieu. Thus, HCK inhibition could represent a novel approach for the treatment of the acute myeloid leukemia.

Tyrosine kinase inhibitor resistance: a case report on chronic myeloid leukemia and Gilbert's syndrome.

Although tyrosine kinase inhibitors (TKI) are commonly used as targeted treatment options for chronic myeloid leukemia (CML), its use is associated to UGT1A1 polymorphisms and, consequently, are related to a higher risk of manifesting Gilbert's syndrome, a genetic disorder associated to hyperbilirubinemia. The report of concomitant condition of CML and Gilbert's Syndrome is uncommon. Therefore, the aim of this study was to report the clinical case of a patient diagnosed with CML and subsequently, with Gilbert's Syndrome. A 34-year-old female was diagnosed with CML. On physical examination, spleen and liver were palpable, indicating hepatosplenomegaly. Laboratory findings of peripheral blood showed leukocytosis (165,190/mm3), 6% of blasts and a bone marrow biopsy showed hypercellularity by granulocytic series with moderate maturation delay. After diagnosis, the patient immediately started chemotherapy with the TKI Imatinib. One year after treatment, due to the partial response, the therapy was changed to Nilotinib, resulting in a complete response. Despite the absence of hyperbilirubinemia, a genetic study by polymerase chain reaction (PCR) verified a positivity for Gilbert's Syndrome. TKIs are also inhibitors of the enzyme UDPGT1, leading to deficient glucuronidation, causing manifestation of Gilbert's Syndrome. This report demonstrates the case of a patient that, besides having two coexisting conditions that could cause hyperbilirubinemia, did not have bilirubin alterations and it highlights the importance of having genetic investigations in cancer patients, in order to identify secondary diseases that could worsen the course of treatment.