RAS-mutant leukaemia stem cells drive clinical resistance to venetoclax.

Cancer driver mutations often show distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in the course of acute myeloid leukaemia, upon progression or relapsed/refractory disease1-6. Here, by using human leukaemogenesis models, we first show that RAS mutations are obligatory late events that need to succeed earlier cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed progenitors of the myelomonocytic lineage (granulocyte-monocyte progenitors) harbouring previously acquired driver mutations, showing that advanced leukaemic clones can originate from a different cell type in the haematopoietic hierarchy than ancestral clones. Furthermore, we demonstrate that RAS-mutant leukaemia stem cells (LSCs) give rise to monocytic disease, as observed frequently in patients with poor responses to treatment with the BCL2 inhibitor venetoclax. We show that this is because RAS-mutant LSCs, in contrast to RAS-wild-type LSCs, have altered BCL2 family gene expression and are resistant to venetoclax, driving clinical resistance and relapse with monocytic features. Our findings demonstrate that a specific genetic driver shapes the non-genetic cellular hierarchy of acute myeloid leukaemia by imposing a specific LSC target cell restriction and critically affects therapeutic outcomes in patients.

BCR::ABL1 Proteolysis-targeting chimeras (PROTACs): The new frontier in the treatment of Ph+ leukemias?

BCR::ABL1 tyrosine kinase inhibitors (TKIs) have turned chronic myeloid leukemia (CML) from a lethal condition into a chronic ailment. With optimal management, the survival of CML patients diagnosed in the chronic phase is approaching that of age-matched controls. However, only one-third of patients can discontinue TKIs and enter a state of functional cure termed treatment-free remission (TFR), while the remainder require life-long TKI therapy to avoid the recurrence of active leukemia. Approximately 10% of patients exhibit primary or acquired TKI resistance and eventually progress to the blast phase. It is thought that recurrence after attempted TFR originates from CML stem cells (LSCs) surviving despite continued suppression of BCR::ABL1 kinase. Although kinase activity is indispensable for induction of overt CML, kinase-independent scaffold functions of BCR::ABL1 are known to contribute to leukemogenesis, raising the intriguing but as yet hypothetical possibility, that degradation of BCR::ABL1 protein may accomplish what TKIs fail to achieve - eliminate residual LSCs to turn functional into real cures. The advent of BCR::ABL1 proteolysis targeting chimeras (PROTACs), heterobifunctional molecules linking a TKI-based warhead to an E3 ligase recruiter, has moved clinical protein degradation into the realm of the possible. Here we examine the molecular rationale as well as pros and cons of degrading BCR::ABL1 protein. We review reported BCR::ABL1 PROTACs, point out limitations of available data and compounds and suggest directions for future research. Ultimately, clinical testing of a potent and specific BCR::ABL1 degrader will be required to determine the efficacy and tolerability of this approach.

MIR4435-2HG as a possible novel predictive biomarker of chemotherapy response and death in pediatric B-cell ALL.

Introduction: Although B-cell acute lymphoblastic leukemia (B-cell ALL) survival rates have improved in recent years, Hispanic children continue to have poorer survival rates. There are few tools available to identify at the time of diagnosis whether the patient will respond to induction therapy. Our goal was to identify predictive biomarkers of treatment response, which could also serve as prognostic biomarkers of death, by identifying methylated and differentially expressed genes between patients with positive minimal residual disease (MRD+) and negative minimal residual disease (MRD-). Methods: DNA and RNA were extracted from tumor blasts separated by immunomagnetic columns. Illumina MethlationEPIC and mRNA sequencing assays were performed on 13 bone marrows from Hispanic children with B-cell ALL. Partek Flow was used for transcript mapping and quantification, followed by differential expression analysis using DEseq2. DNA methylation analyses were performed with Partek Genomic Suite and Genome Studio. Gene expression and differential methylation were compared between patients with MRD-/- and MRD+/+ at the end of induction chemotherapy. Overexpressed and hypomethylated genes were selected and validated by RT-qPCR in samples of an independent validation cohort. The predictive ability of the genes was assessed by logistic regression. Survival and Cox regression analyses were performed to determine the association of genes with death. Results: DAPK1, BOC, CNKSR3, MIR4435-2HG, CTHRC1, NPDC1, SLC45A3, ITGA6, and ASCL2 were overexpressed and hypomethylated in MRD+/+ patients. Overexpression was also validated by RT-qPCR. DAPK1, BOC, ASCL2, and CNKSR3 can predict refractoriness, but MIR4435-2HG is the best predictor. Additionally, higher expression of MIR4435-2HG increases the probability of non-response, death, and the risk of death. Finally, MIR4435-2HG overexpression, together with MRD+, are associated with poorer survival, and together with overexpression of DAPK1 and ASCL2, it could improve the risk classification of patients with normal karyotype. Conclusion: MIR4435-2HG is a potential predictive biomarker of treatment response and death in children with B-cell ALL.

Plant-derived extracts and metabolic modulation in leukemia: a promising approach to overcome treatment resistance.

Leukemic cells acquire complex and often multifactorial mechanisms of resistance to treatment, including various metabolic alterations. Although the use of metabolic modulators has been proposed for several decades, their use in clinical practice has not been established. Natural products, the so-called botanical drugs, are capable of regulating tumor metabolism, particularly in hematopoietic tumors, which could partly explain the biological activity attributed to them for a long time. This review addresses the most recent findings relating to metabolic reprogramming-Mainly in the glycolytic pathway and mitochondrial activity-Of leukemic cells and its role in the generation of resistance to conventional treatments, the modulation of the tumor microenvironment, and the evasion of immune response. In turn, it describes how the modulation of metabolism by plant-derived extracts can counteract resistance to chemotherapy in this tumor model and contribute to the activation of the antitumor immune system.

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. SIGNIFICANCE: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov, p. 252. This article is highlighted in the In This Issue feature, p. 247.

Mecanismos moleculares emergentes y células madre leucémicas en la quimiorresistencia de tumores hematológicos / Emerging molecular mechanisms and leukemic stem cells in chemoresistance of hematologic tumors

Resumen Las leucemias agudas son trastornos clonales originados a partir de células hematopoyéticas primitivas multipotenciales que se caracterizan por la proliferación, diferenciación y maduración aberrante de células progenitoras leucémicas como resultado de varios eventos genéticos y epigenéticos. Aunque en la actualidad se han implementado diferentes esquemas de quimioterapia para mejorar el pronóstico de los pacientes, las leucemias agudas representan una malignidad hematológica con pobre desenlace clínico y bajas tasas de supervivencia en pacientes pediátricos y adultos Colombianos. Uno de los principales obstáculos para el tratamiento exitoso del cáncer es el desarrollo de resistencia a los medicamentos durante la quimioterapia y la enfermedad recurrente. En el estudio de la biología de las células tumorales, se reconoce que los diversos cambios oncogénicos y la evolución clonal que sufren las células tumorales, son cambios biológicos que les confieren mecanismos de resistencia a la quimioterapia convencional, que a su vez se traducen en un incremento en las tasas de mortalidad y/o el aumento de recaídas en los pacientes que padecen esta enfermedad. Por lo tanto, el estudio de los mecanismos empleados por las células leucémicas para escapar del efecto citotóxico del tratamiento empleado para combatir la enfermedad es un objetivo primordial de la investigación en cáncer. En este contexto, el objetivo del presente artículo es hacer una revisión detallada de los avances más recientes en la comprensión de los mecanismos involucrados en la resistencia tumoral en leucemias, haciendo especial énfasis en el papel que desempeñan las células madre leucémicas y el metabolismo tumoral en la quimiorresistencia de este grupo de enfermedades. El conocimiento de los mecanismos de resistencia tumoral, así como el entendimiento detallado de las interacciones entre las células normales y leucémicas en el microambiente de la médula ósea, son prometedores blancos terapéuticos de las leucemias agudas.

Abstract Acute leukemias (AL) are clonal disorders originated from multi-potent immature hematopoietic cells and are characterized by aberrant proliferation, differentiation and maturation of leukemic progenitor cells as a result of multiple genetic and epigenetic events. Even though different chemotherapy regimens have been implemented to improve patient prognostic, acute leukemias represent a hematological malignancy with poor clinical outcome and low survival rates in pediatric and adult patients in Colombia. One of the main obstacles to the success of cancer treatment is the development of drug resistance during chemotherapy and the recurrent disease. In the study of tumor cells biology, it is now known that clonal evolution and oncogenic changes of tumor cells are biological properties that confer resistance mechanisms to conventional chemotherapy, which in turn translate into an increased mortality rate and/or an increased risk of relapse in leukemia patients. Therefore, the study of mechanisms that leukemic cells employ to avoid the cytotoxic effects of some chemotherapeutics is a main objective of cancer research. In this context, the objective of the current paper is to give a detailed information about recent advances in mechanisms involved in leukemic resistance, with special emphasis on the role of leukemic stem cells theory and tumor metabolism.

Establishment of Two Dimensional (2D) and Three-Dimensional (3D) Melanoma Primary Cultures as a Tool for In Vitro Drug Resistance Studies.

Characteristics of melanoma cells have been deciphered by studies carried out in two dimensional cell cultures growing as adherent monolayers on the bottom of plastic flasks. Melanoma cells can be cultured with a considerable degree of success, and, depending on the further use of the cells obtained in the culture, methodologies have to be adjusted to obtain reliable results. Although there are many melanoma continuous cell lines, in vitro 2D and 3D melanoma primary cell culture may be a more useful model to investigate interactions between cancer cells and immune system, as well as the effect of cytotoxic treatments and personalized medicine in environments more similar to the physiological conditions.Here, we described a protocol which employs many strategies to obtain primary 2D and 3D melanoma cultures as a model to study cell-cell and cell-microenvironment interactions that must be considered to properly design personalized cancer treatments, as well as for testing novel anticancer drugs and drug delivery vehicles.

Epigenetics in Hematological Malignancies.

Acute leukemias are hematologic malignancies with aggressive behavior especially in adult population. With the introduction of new gene expression and sequencing technologies there have been advances in the knowledge of the genetic landscape of acute leukemias. A more detailed analysis allows for the identification of additional alterations in epigenetic regulators that have a profound impact in cellular biology without changes in DNA sequence. These epigenetic alterations disturb the physiological balance between gene activation and gene repression and contribute to aberrant gene expression, contributing significantly to the leukemic pathogenesis and maintenance. We review epigenetic changes in acute leukemia in relation to what is known about their mechanism of action, their prognostic role and their potential use as therapeutic targets, with important implications for precision medicine.

Increased expression of deleted in malignant brain tumors (DMBT1) gene in precancerous gastric lesions: Findings from human and animal studies.

Helicobacter pylori infection triggers a cascade of inflammatory stages that may lead to the appearance of non-atrophic gastritis, multifocal atrophic, intestinal metaplasia, dysplasia, and cancer. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by binding to pathogens. Initial studies showed its deletion and loss of expression in a variety of tumors but the role of this gene in tumor development is not completely understood. Here, we examined the role of DMBT1 in gastric precancerous lesions in Caucasian, African American and Hispanic individuals as well as in the development of gastric pathology in a mouse model of H. pylori infection. We found that in 3 different populations, mucosal DMBT1 expression was significantly increased (2.5 fold) in individuals with dysplasia compared to multifocal atrophic gastritis without intestinal metaplasia; the increase was also observed in individuals with advanced gastritis and positive H. pylori infection. In our animal model, H. pylori infection of Dmbt1-/- mice resulted in significantly higher levels of gastritis, more extensive mucous metaplasia and reduced Il33 expression levels in the gastric mucosa compared to H. pylori-infected wild type mice. Our data in the animal model suggest that in response to H. pylori infection DMBT1 may mediate mucosal protection reducing the risk of developing gastric precancerous lesions. However, the increased expression in human gastric precancerous lesions points to a more complex role of DMBT1 in gastric carcinogenesis.

Prognostic stratification improvement by integrating ID1/ID3/IGJ gene expression signature and immunophenotypic profile in adult patients with B-ALL.

BACKGROUND: Survival of adults with B-Acute Lymphoblastic Leukemia requires accurate risk stratification of patients in order to provide the appropriate therapy. Contemporary techniques, using clinical and cytogenetic variables are incomplete for prognosis prediction. METHODS: To improve the classification of adult patients diagnosed with B-ALL into prognosis groups, two strategies were examined and combined: the expression of the ID1/ID3/IGJ gene signature by RT-PCR and the immunophenotypic profile of 19 markers proposed in the EuroFlow protocol by Flow Cytometry in bone marrow samples. RESULTS: Both techniques were correlated to stratify patients into prognostic groups. An inverse relationship between survival and expression of the three-genes signature was observed and an immunophenotypic profile associated with clinical outcome was identified. Markers CD10 and CD20 were correlated with simultaneous overexpression of ID1, ID3 and IGJ. Patients with simultaneous expression of the poor prognosis gene signature and overexpression of CD10 or CD20, had worse Event Free Survival and Overall Survival than patients who had either the poor prognosis gene expression signature or only CD20 or CD10 overexpressed. CONCLUSION: By utilizing the combined evaluation of these two immunophenotypic markers along with the poor prognosis gene expression signature, the risk stratification can be significantly strengthened. Further studies including a large number of patients are needed to confirm these findings.

High expression of ID family and IGJ genes signature as predictor of low induction treatment response and worst survival in adult Hispanic patients with B-acute lymphoblastic leukemia.

BACKGROUND: B-Acute lymphoblastic leukemia (B-ALL) represents a hematologic malignancy with poor clinical outcome and low survival rates in adult patients. Remission rates in Hispanic population are almost 30% lower and Overall Survival (OS) nearly two years inferior than those reported in other ethnic groups. Only 61% of Colombian adult patients with ALL achieve complete remission (CR), median overall survival is 11.3 months and event-free survival (EFS) is 7.34 months. Identification of prognostic factors is crucial for the application of proper treatment strategies and subsequently for successful outcome. Our goal was to identify a gene expression signature that might correlate with response to therapy and evaluate the utility of these as prognostic tool in hispanic patients. METHODS: We included 43 adult patients newly diagnosed with B-ALL. We used microarray analysis in order to identify genes that distinguish poor from good response to treatment using differential gene expression analysis. The expression profile was validated by real-time PCR (RT-PCT). RESULTS: We identified 442 differentially expressed genes between responders and non-responders to induction treatment. Hierarchical analysis according to the expression of a 7-gene signature revealed 2 subsets of patients that differed in their clinical characteristics and outcome. CONCLUSIONS: Our study suggests that response to induction treatment and clinical outcome of Hispanic patients can be predicted from the onset of the disease and that gene expression profiles can be used to stratify patient risk adequately and accurately. The present study represents the first that shows the gene expression profiling of B-ALL Colombian adults and its relevance for stratification in the early course of disease.