Resistance Mechanisms in Pediatric B-Cell Acute Lymphoblastic Leukemia

Despite the rapid development of medicine, even nowadays, acute lymphoblastic leukemia (ALL) is still a problem for pediatric clinicians. Modern medicine has reached a limit of curability even though the recovery rate exceeds 90%. Relapse occurs in around 20% of treated patients and, regrettably, 10% of diagnosed ALL patients are still incurable. In this article, we would like to focus on the treatment resistance and disease relapse of patients with B-cell leukemia in the context of prognostic factors of ALL. We demonstrate the mechanisms of the resistance to steroid therapy and Tyrosine Kinase Inhibitors and assess the impact of genetic factors on the treatment resistance, especially TCF3::HLF translocation. We compare therapeutic protocols and decipher how cancer cells become resistant to innovative treatments—including CAR-T-cell therapies and monoclonal antibodies. The comparisons made in our article help to bring closer the main factors of resistance in hematologic malignancies in the context of ALL.     

The Bone Marrow Niche in B-Cell Acute Lymphoblastic Leukemia: The Role of Microenvironment from Pre-Leukemia to Overt Leukemia

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a “corrupted” BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.     

Axitinib in Ponatinib-Resistant B-Cell Acute Lymphoblastic Leukemia Harboring a T315L Mutation

Adult acute lymphoblastic leukemia (ALL) with BCR-ABL1 rearrangement (Philadelphia chromosome, Ph) is a hematological aggressive disease with a fatal outcome in more than 50% of cases. Tyrosine kinase inhibitors (TKIs) targeting the activity of BCR-ABL1 protein have improved the prognosis; however, relapses are frequent because of acquired somatic mutations in the BCR-ABL1 kinase domain causing resistance to first, second and third generation TKIs. Axitinib has shown in vitro and ex vivo activity in blocking ABL1; however, clinical trials exploring its efficacy in ALL are missing. Here, we presented a 77-year-old male with a diagnosis of Ph positive ALL resistant to ponatinib and carrying a rare threonine to leucine (T315L) mutation on BCR-ABL1 gene. The patient was treated with axitinib at 5 mg/twice daily as salvage therapy showing an immediate although transient benefit with an overall survival of 9.3 months. Further dose-finding and randomized clinical trials are required to assess the real efficacy of axitinib for adult Ph positive ALL resistant to third generation TKIs.     

Genetic Biomarkers and Their Clinical Implications in B-Cell Acute Lymphoblastic Leukemia in Children

Acute lymphoblastic leukemia (ALL) is a heterogeneous group of hematologic malignancies characterized by abnormal proliferation of immature lymphoid cells. It is the most commonly diagnosed childhood cancer with an almost 80% cure rate. Despite favorable survival rates in the pediatric population, a significant number of patients develop resistance to therapy, resulting in poor prognosis. ALL is a heterogeneous disease at the genetic level, but the intensive development of sequencing in the last decade has made it possible to broaden the study of genomic changes. New technologies allow us to detect molecular changes such as point mutations or to characterize epigenetic or proteomic profiles. This process made it possible to identify new subtypes of this disease characterized by constellations of genetic alterations, including chromosome changes, sequence mutations, and DNA copy number alterations. These genetic abnormalities are used as diagnostic, prognostic and predictive biomarkers that play an important role in earlier disease detection, more accurate risk stratification, and treatment. Identification of new ALL biomarkers, and thus a greater understanding of their molecular basis, will lead to better monitoring of the course of the disease. In this article, we provide an overview of the latest information on genomic alterations found in childhood ALL and discuss their impact on patients’ clinical outcomes.     

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated β-Galactosidase (SA-βGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-βGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.     

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.     

Galectin-1 and Galectin-3 in B-Cell Precursor Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemias arising from the malignant transformation of B-cell precursors (BCP-ALLs) are protected against chemotherapy by both intrinsic factors as well as by interactions with bone marrow stromal cells. Galectin-1 and Galectin-3 are lectins with overlapping specificity for binding polyLacNAc glycans. Both are expressed by bone marrow stromal cells and by hematopoietic cells but show different patterns of expression, with Galectin-3 dynamically regulated by extrinsic factors such as chemotherapy. In a comparison of Galectin-1 × Galectin-3 double null mutant to wild-type murine BCP-ALL cells, we found reduced migration, inhibition of proliferation, and increased sensitivity to drug treatment in the double knockout cells. Plant-derived carbohydrates GM-CT-01 and GR-MD-02 were used to inhibit extracellular Galectin-1/-3 binding to BCP-ALL cells in co-culture with stromal cells. Treatment with these compounds attenuated migration of the BCP-ALL cells to stromal cells and sensitized human BCP-ALL cells to vincristine and the targeted tyrosine kinase inhibitor nilotinib. Because N-glycan sialylation catalyzed by the enzyme ST6Gal1 can regulate Galectin cell-surface binding, we also compared the ability of BCP-ALL wild-type and ST6Gal1 knockdown cells to resist vincristine treatment when they were co-cultured with Galectin-1 or Galectin-3 knockout stromal cells. Consistent with previous results, stromal Galectin-3 was important for maintaining BCP-ALL fitness during chemotherapy exposure. In contrast, stromal Galectin-1 did not significantly contribute to drug resistance, and there was no clear effect of ST6Gal1-catalysed N-glycan sialylation. Taken together, our results indicate a complicated joint contribution of Galectin-1 and Galectin-3 to BCP-ALL survival, with different roles for endogenous and stromal produced Galectins. These data indicate it will be important to efficiently block both extracellular and intracellular Galectin-1 and Galectin-3 with the goal of reducing BCP-ALL persistence in the protective bone marrow niche during chemotherapy.     

Non-Coding RNAs and Cancer

The discovery of the biological relevance of non-coding RNA (ncRNAs) molecules represents one of the most significant advances in contemporary molecular biology. Expression profiling of human tumors, based on the expression of miRNAs and other short or long ncRNAs, has identified signatures associated with diagnosis, staging, progression, prognosis, and response to treatment. In this review we will discuss the recent remarkable advancement in the understanding the biological functions of human ncRNAs in cancer, the mechanisms of expression and the therapeutic potential.     

Epigenetic Control of Infant B Cell Precursor Acute Lymphoblastic Leukemia

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a highly aggressive malignancy, with poorer prognosis in infants than in adults. A genetic signature has been associated with this outcome but, remarkably, leukemogenesis is commonly triggered by genetic alterations of embryonic origin that involve the deregulation of chromatin remodelers. This review considers in depth how the alteration of epigenetic profiles (at DNA and histone levels) induces an aberrant phenotype in B lymphocyte progenitors by modulating the oncogenic drivers and tumor suppressors involved in key cancer hallmarks. DNA methylation patterns have been widely studied in BCP-ALL and their correlation with survival has been established. However, the effect of methylation on histone residues can be very different. For instance, methyltransferase KMT2A gene participates in chromosomal rearrangements with several partners, imposing an altered pattern of methylated H3K4 and H3K79 residues, enhancing oncogene promoter activation, and conferring a worse outcome on affected infants. In parallel, acetylation processes provide an additional layer of epigenetic regulation and can alter the chromatin conformation, enabling the binding of regulatory factors. Therefore, an integrated knowledge of all epigenetic disorders is essential to understand the molecular basis of BCP-ALL and to identify novel entry points that can be exploited to improve therapeutic options and disease prognosis.     

The Emerging Role of Venetoclax-Based Treatments in Acute Lymphoblastic Leukemia

Venetoclax, a B-cell lymphoma (BCL-2) inhibitor, in combination with hypomethylating agents has become the new standard of care in elderly and unfit patients with acute myeloid leukemia, with significantly improved overall survival and quality of life. Studies of venetoclax combined with high-dose chemotherapy are emerging with evidence of higher rates of molecular remission. Recently, a growing number of publications bring forth the use of venetoclax in patients with acute lymphoblastic leukemia (ALL). In the current review, we present the biological rationale of BCL-2 inhibition in ALL, how the interplay of BH3 proteins modulate the response and the current clinical experience with various combinations.     

Targeting Telomere Biology in Acute Lymphoblastic Leukemia

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target; hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.     

Neurotoxicity Associated with Treatment of Acute Lymphoblastic Leukemia Chemotherapy and Immunotherapy

Immunotherapy is a milestone in the treatment of poor-prognosis pediatric acute lymphoblastic leukemia (ALL) and is expected to improve treatment outcomes and reduce doses of conventional chemotherapy without compromising the effectiveness of the therapy. However, both chemotherapy and immunotherapy cause side effects, including neurological ones. Acute neurological complications occur in 3.6–11% of children treated for ALL. The most neurotoxical chemotherapeutics are L-asparaginase (L-ASP), methotrexate (MTX), vincristine (VCR), and nelarabine (Ara-G). Neurotoxicity associated with methotrexate (MTX-NT) occurs in 3–7% of children treated for ALL and is characterized by seizures, stroke-like symptoms, speech disturbances, and encephalopathy. Recent studies indicate that specific polymorphisms in genes related to neurogenesis may have a predisposition to MTX toxicity. One of the most common complications associated with CAR T-cell therapy is immune effector cell-associated neurotoxicity syndrome (ICANS). Mechanisms of neurotoxicity in CAR T-cell therapy are still unknown and may be due to disruption of the blood–brain barrier and the effects of elevated cytokine levels on the central nervous system (CNS). In this review, we present an analysis of the current knowledge on the mechanisms of neurotoxicity of standard chemotherapy and the targeted therapy in children with ALL.     

An Analysis of Differentially Expressed Coding and Long Non-Coding RNAs in Multiple Models of Skeletal Muscle Atrophy

The loss of skeletal muscle mass (muscle atrophy or wasting) caused by aging, diseases, and injury decreases quality of life, survival rates, and healthy life expectancy in humans. Although long non-coding RNAs (lncRNAs) have been implicated in skeletal muscle formation and differentiation, their precise roles in muscle atrophy remain unclear. In this study, we used RNA-sequencing (RNA-Seq) to examine changes in the expression of lncRNAs in four muscle atrophy conditions (denervation, casting, fasting, and cancer cachexia) in mice. We successfully identified 33 annotated lncRNAs and 18 novel lncRNAs with common expression changes in all four muscle atrophy conditions. Furthermore, an analysis of lncRNA–mRNA correlations revealed that several lncRNAs affected small molecule biosynthetic processes during muscle atrophy. These results provide novel insights into the lncRNA-mediated regulatory mechanism underlying muscle atrophy and may be useful for the identification of promising therapeutic targets.     

A Novel Triple-Action Inhibitor Targeting B-Cell Receptor Signaling and BRD4 Demonstrates Preclinical Activity in Chronic Lymphocytic Leukemia

B-cell chronic lymphocytic leukemia (CLL) results from intrinsic genetic defects and complex microenvironment stimuli that fuel CLL cell growth through an array of survival signaling pathways. Novel small-molecule agents targeting the B-cell receptor pathway and anti-apoptotic proteins alone or in combination have revolutionized the management of CLL, yet combination therapy carries significant toxicity and CLL remains incurable due to residual disease and relapse. Single-molecule inhibitors that can target multiple disease-driving factors are thus an attractive approach to combat both drug resistance and combination-therapy-related toxicities. We demonstrate that SRX3305, a novel small-molecule BTK/PI3K/BRD4 inhibitor that targets three distinctive facets of CLL biology, attenuates CLL cell proliferation and promotes apoptosis in a dose-dependent fashion. SRX3305 also inhibits the activation-induced proliferation of primary CLL cells in vitro and effectively blocks microenvironment-mediated survival signals, including stromal cell contact. Furthermore, SRX3305 blocks CLL cell migration toward CXCL-12 and CXCL-13, which are major chemokines involved in CLL cell homing and retention in microenvironment niches. Importantly, SRX3305 maintains its anti-tumor effects in ibrutinib-resistant CLL cells. Collectively, this study establishes the preclinical efficacy of SRX3305 in CLL, providing significant rationale for its development as a therapeutic agent for CLL and related disorders.