p21-Activated kinases as promising therapeutic targets in hematological malignancies.

The p21-Activated Kinases (PAKs) are a family of six serine/threonine kinases that were originally identified as downstream effectors of the Rho GTPases Cdc42 and Rac. Since the first PAK was discovered in 1994, studies have revealed their fundamental and biological importance in the development of physiological systems. Within the cell, PAKs also play significant roles in regulating essential cellular processes such as cytoskeletal dynamics, gene expression, cell survival, and cell cycle progression. These processes are often deregulated in numerous cancers when different PAKs are overexpressed or amplified at the chromosomal level. Furthermore, PAKs modulate multiple oncogenic signaling pathways which facilitate apoptosis escape, uncontrolled proliferation, and drug resistance. There is growing insight into the critical roles of PAKs in regulating steady-state hematopoiesis, including the properties of hematopoietic stem cells (HSC), and the initiation and progression of hematological malignancies. This review will focus on the most recent studies that provide experimental evidence showing how specific PAKs regulate the properties of leukemic stem cells (LSCs) and drug-resistant cells to initiate and maintain hematological malignancies. The current understanding of the molecular and cellular mechanisms by which the PAKs operate in specific human leukemia or lymphomas will be discussed. From a translational point of view, PAKs have been suggested to be critical therapeutic targets and potential prognosis markers; thus, this review will also discuss current therapeutic strategies against hematological malignancies using existing small-molecule PAK inhibitors, as well as promising combination treatments, to sensitize drug-resistant cells to conventional therapies. The challenges of toxicity and non-specific targeting associated with some PAK inhibitors, as well as how future approaches for PAK inhibition to overcome these limitations, will also be addressed.

The Use of Inhibitors of Tyrosine Kinase in Paediatric Haemato-Oncology-When and Why?

The fundamental pathophysiology of malignancies is dysregulation of the signalling pathways. Protein tyrosine kinases (PTKs) are among the enzymes which, if mutated, play a critical role in carcinogenesis. The best-studied rearrangement, which enhances PTK activity and causes atypical proliferation, is BCR-ABL1. Abnormal expression of PTKs has proven to play a significant role in the development of various malignancies, such as chronic myelogenous leukaemia, brain tumours, neuroblastoma, and gastrointestinal stromal tumours. The use of tyrosine kinase inhibitors (TKIs) is an outstanding example of successful target therapy. TKIs have been effectively applied in the adult oncology setting, but there is a need to establish TKIs' importance in paediatric patients. Many years of research have allowed a significant improvement in the outcome of childhood cancers. However, there are still groups of patients who have a poor prognosis, where the intensification of chemotherapy could even cause death. TKIs are designed to target specific PTKs, which lead to the limitation of severe adverse effects and increase overall survival. These advances will hopefully allow new therapeutic approaches in paediatric haemato-oncology to emerge. In this review, we present an analysis of the current data on tyrosine kinase inhibitors in childhood cancers.

Immunoproteasome Function in Normal and Malignant Hematopoiesis.

The ubiquitin-proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

Resistance Mutations to BTK Inhibitors Originate From the NF-κB but Not From the PI3K-RAS-MAPK Arm of the B Cell Receptor Signaling Pathway.

Since the first clinical report in 2013, inhibitors of the intracellular kinase BTK (BTKi) have profoundly altered the treatment paradigm of B cell malignancies, replacing chemotherapy with targeted agents in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström's macroglobulinemia. There are over 20 BTKi, both irreversible and reversible, in clinical development. While loss-of-function (LoF) mutations in the BTK gene cause the immunodeficiency X-linked agammaglobulinemia, neither inherited, nor somatic BTK driver mutations are known. Instead, BTKi-sensitive malignancies are addicted to BTK. BTK is activated by upstream surface receptors, especially the B cell receptor (BCR) but also by chemokine receptors, and adhesion molecules regulating B cell homing. Consequently, BTKi therapy abrogates BCR-driven proliferation and the tissue homing capacity of the malignant cells, which are being redistributed into peripheral blood. BTKi resistance can develop over time, especially in MCL and high-risk CLL patients. Frequently, resistance mutations affect the BTKi binding-site, cysteine 481, thereby reducing drug binding. Less common are gain-of-function (GoF) mutations in downstream signaling components, including phospholipase Cγ2 (PLCγ2). In a subset of patients, mechanisms outside of the BCR pathway, related e.g. to resistance to apoptosis were described. BCR signaling depends on many proteins including SYK, BTK, PI3K; still based on the resistance pattern, BTKi therapy only selects GoF alterations in the NF-κB arm, whereas an inhibitor of the p110δ subunit of PI3K instead selects resistance mutations in the RAS-MAP kinase pathway. BTK and PLCγ2 resistance mutations highlight BTK's non-redundant role in BCR-mediated NF-κB activation. Of note, mutations affecting BTK tend to generate clone sizes larger than alterations in PLCγ2. This infers that BTK signaling may go beyond the PLCγ2-regulated NF-κB and NFAT arms. Collectively, when comparing the primary and acquired mutation spectrum in BTKi-sensitive malignancies with the phenotype of the corresponding germline alterations, we find that certain observations do not readily fit with the existing models of BCR signaling.

Inhibitors of gelatinases (MMP-2 and MMP-9) for the management of hematological malignancies.

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are collectively known as gelatinases whereas MMP-2 is gelatinase-A and MMP-9 is termed as gelatinase-B. Gelatinases and other matrix metalloproteinases (MMPs) have long been associated with solid tumor invasion, metastasis and angiogenesis. However, there is paucity of data available regarding the role of gelatinases in hematological malignancies. Recent studies have shown that gelatinases activities or functions are correlated with hematological malignancies. Strategies for designing more specific gelatinase inhibitors like catalytic (CAT) domain inhibitors and hemopexin (PEX) domain inhibitors as well as signaling pathway based or gelatinase expression inhibitors had been reported against hematologic malignant cells. Several substrate based non-selective to non-substrate based relatively selective synthetic matrix metalloproteinase inhibitors (MMPIs) had been developed. Few MMPIs had reached in clinical trials during the period of 1990s-2000s. Unfortunately the anti-tumor and anti-metastatic efficacies of these MMPIs were not justified with patients having several advanced stage solid tumor cancers in any substantial number of clinical trials. Till date not a single MMPI passed phase III clinical trials designed for advanced metastatic cancers due to adverse events as well as lack of ability to show uniformity in disease prolongation. With the best of our knowledge no clinical trial study has been reported with small molecule synthetic inhibitors against hematological malignancies. This review looks at the outcome of clinical trials of MMPIs for advanced stage solid tumors. This can therefore, act as a learning experience for future development of successful gelatinase inhibitors for the management of hematological malignancies.

JAK2V617F mutant endothelial cells promote neoplastic hematopoiesis in a mixed vascular microenvironment.

The chronic myeloproliferative neoplasms (MPNs) are clonal stem cell disorders. The hematopoietic stem/progenitor cell (HSPC) compartment in patients with MPNs is heterogeneous with the presence of both wild-type and JAK2V617F mutant cells. Mechanisms responsible for mutant stem cell expansion in MPNs are not fully understood. Vascular endothelial cells (ECs) are an essential component of the hematopoietic microenvironment. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Utilizing an ex vivo EC-HSPC co-culture system with mixed wild-type and JAK2V617F mutant ECs, we show that even small numbers of JAK2V617F mutant ECs can promote the expansion of JAK2V617F mutant HSPCs in preference to wild-type HSPCs during irradiation or cytotoxic chemotherapy, the two treatments commonly used in the conditioning regimen for stem cell transplantation, the only curative treatment for patients with MPNs. Mechanistically, we found that both cell-cell interactions and secreted factors are important for JAK2V617F mutant EC-mediated neoplastic hematopoiesis. Further understanding of how the JAK2V617F mutation alters vascular niche function will help identify new strategies to not only control neoplastic cell expansion but also prevent disease relapse in patients with MPNs.

PI3 kinase signaling pathway in hematopoietic cancers: A glance in miRNA's role.

Hematopoietic cancers are among the most common malignancies worldwide, which are divided into different types depending on the origin of tumor cells. In recent years, the pivotal role of different signaling pathways in the onset and progression of these cancer types has been well established. One of these pathways, whose role in blood malignancies has been well-defined, is PI3K/mTOR/AKT axis. The signaling pathway involves in a wide variety of important biological events in cells. It is clear that dysregulation of mediators involved in PI3 kinase signaling takes a pivotal role in cancer development. Considering the undeniable role of miRNAs, as one of the well-known families of non-coding RNAs, in gene regulation, we aimed to review the role of miRNAs in regulation of PI3 kinase signaling effectors in hematopoietic cancers.

ADARs, RNA editing and more in hematological malignancies.

Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in humans, mediated by the adenosine deaminases acting on RNA (ADARs). Physiologically, these enzymes are present in the nucleus and/or the cytoplasm, where they catalyze the conversion of adenosines (A) to inosines (I) on double-stranded mRNA molecules. Aberrant ADAR-mediated-editing is a prominent feature in a variety of cancers. Importantly, the biological functions of ADARs and its functional implications in hematological malignancies have recently been unraveled. In this review, we will highlight the functions of ADARs and their involvements in cancer, specifically in hematological malignancies. RNA editing-independent function of cellular processes by ADARs and the potential of developing novel therapeutic approaches revolving RNA editing will also be discussed.

Molecular and clinical features of myeloid neoplasms with somatic DDX41 mutations.

We screened 47 subjects with DDX41 variants among 1529 subjects with myeloid neoplasms. The most common germline variants included Splice c.935 + 4A>T, p.T360Ifs*33, p.V152G, p.S217Ifs*4, p.R311* and p.R369*. Except for the p.R369*, no other variants have been previously reported. Clinical covariates of subjects with simple DDX41 somatic variants and germline/somatic biallelic variants are similar. The two-year overall survival (OS) of subjects with DDX41 variants was 85%. Overall response rate to demethylation therapy in subjects with DDX41 variants was 69%. The response did not correlate with the presence of a germline variant.

Targeting Casein Kinase 1 (CK1) in Hematological Cancers.

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.

A WEE1 family business: regulation of mitosis, cancer progression, and therapeutic target.

The inhibition of the DNA damage response (DDR) pathway in the treatment of cancer has recently gained interest, and different DDR inhibitors have been developed. Among them, the most promising ones target the WEE1 kinase family, which has a crucial role in cell cycle regulation and DNA damage identification and repair in both nonmalignant and cancer cells. This review recapitulates and discusses the most recent findings on the biological function of WEE1/PKMYT1 during the cell cycle and in the DNA damage repair, with a focus on their dual role as tumor suppressors in nonmalignant cells and pseudo-oncogenes in cancer cells. We here report the available data on the molecular and functional alterations of WEE1/PKMYT1 kinases in both hematological and solid tumors. Moreover, we summarize the preclinical information on 36 chemo/radiotherapy agents, and in particular their effect on cell cycle checkpoints and on the cellular WEE1/PKMYT1-dependent response. Finally, this review outlines the most important pre-clinical and clinical data available on the efficacy of WEE1/PKMYT1 inhibitors in monotherapy and in combination with chemo/radiotherapy agents or with other selective inhibitors currently used or under evaluation for the treatment of cancer patients.

Response to tyrosine kinase inhibitors in myeloid neoplasms associated with PCM1-JAK2, BCR-JAK2 and ETV6-ABL1 fusion genes.

We report on 18 patients with myeloid neoplasms and associated tyrosine kinase (TK) fusion genes on treatment with the TK inhibitors (TKI) ruxolitinib (PCM1-JAK2, n = 8; BCR-JAK2, n = 1) and imatinib, nilotinib or dasatinib (ETV6-ABL1, n = 9). On ruxolitinib (median 24 months, range 2-36 months), a complete hematologic response (CHR) and complete cytogenetic response (CCR) was achieved by five of nine and two of nine patients, respectively. However, ruxolitinib was stopped in eight of nine patients because of primary resistance (n = 3), progression (n = 3) or planned allogeneic stem cell transplantation (allo SCT, n = 2). At a median of 36 months (range 4-78 months) from diagnosis, five of nine patients are alive: four of six patients after allo SCT and one patient who remains on ruxolitinib. In ETV6-ABL1 positive patients, a durable CHR was achieved by four of nine patients (imatinib with one of five, nilotinib with two of three, dasatinib with one of one). Because of inadequate efficacy (lack of hematological and/or cytogenetic/molecular response), six of nine patients (imatinib, n = 5; nilotinib, n = 1) were switched to nilotinib or dasatinib. At a median of 23 months (range 3-60 months) from diagnosis, five of nine patients are in CCR or complete molecular response (nilotinib, n = 2; dasatinib, n = 2; allo SCT, n = 1) while two of nine patients have died. We conclude that (a) responses on ruxolitinib may only be transient in the majority of JAK2 fusion gene positive patients with allo SCT being an important early treatment option, and (b) nilotinib or dasatinib may be more effective than imatinib to induce durable complete remissions in ETV6-ABL1 positive patients.

Deubiquitinases: Pro-oncogenic Activity and Therapeutic Targeting in Blood Malignancies.

Deubiquitinases are enzymes that remove ubiquitin moieties from the vast majority of cellular proteins, controlling their stability, interactions, and localization. The expression and activity of deubiquitinases are critical for physiology and can go awry in various diseases, including cancer. Based on recent findings in human blood cancers, we discuss the functions of selected deubiquitinases in acute leukemia and efforts to target these enzymes with the aim of blocking leukemia growth and improving disease outcomes. We focus on the emergence of the newest generation of preclinical inhibitors by discussing their modes of inhibition and their effects on leukemia biology.

Ectonucleotidases in Blood Malignancies: A Tale of Surface Markers and Therapeutic Targets.

Leukemia develops as the result of intrinsic features of the transformed cell, such as gene mutations and derived oncogenic signaling, and extrinsic factors, such as a tumor-friendly, immunosuppressed microenvironment, predominantly in the lymph nodes and the bone marrow. There, high extracellular levels of nucleotides, mainly NAD+ and ATP, are catabolized by different ectonucleotidases, which can be divided in two families according to substrate specificity: on one side those that metabolize NAD+, including CD38, CD157, and CD203a; on the other, those that convert ATP, namely CD39 (and other ENTPDases) and CD73. They generate products that modulate intracellular calcium levels and that activate purinergic receptors. They can also converge on adenosine generation with profound effects, both on leukemic cells, enhancing chemoresistance and homing, and on non-malignant immune cells, polarizing them toward tolerance. This review will first provide an overview of ectonucleotidases expression within the immune system, in physiological and pathological conditions. We will then focus on different hematological malignancies, discussing their role as disease markers and possibly pathogenic agents. Lastly, we will describe current efforts aimed at therapeutic targeting of this family of enzymes.

DUBbing Down Translation: The Functional Interaction of Deubiquitinases with the Translational Machinery.

Cancer cells revamp the regulatory processes that control translation to induce tumor-specific translational programs that can adapt to a hostile microenvironment as well as withstand anticancer therapeutics. Translational initiation has been established as a common downstream effector of numerous deregulated signaling pathways that together culminate in prooncogenic expression. Other mechanisms, including ribosomal stalling and stress granule assembly, also appear to be rewired in the malignant phenotype. Therefore, better understanding of the underlying perturbations driving oncogenic translation in the transformed state will provide innovative therapeutic opportunities. This review highlights deubiquitinating enzymes that are activated/dysregulated in hematologic malignancies, thereby altering the translational output and contributing to tumorigenesis.

Dysregulation of the TET family of epigenetic regulators in lymphoid and myeloid malignancies.

DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting, X-chromosome inactivation, and cancer. Cancer cells display highly dysregulated DNA methylation profiles, characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands; these changes are often correlated with promoter hypermethylation, leading to decreased expression of tumor suppressor genes, as well as with genome instability, leading to amplification and aberrant expression of oncogenes. Ten-eleven-translocation (TET) proteins are α-ketoglutarate (α-KG)-dependent dioxygenases that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and the additional oxidation products 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC); together, these oxidized methylcytosines are intermediates in DNA demethylation. TET2 is frequently mutated in diverse lymphoid and myeloid cancers, and TET loss of function is often observed in the absence of coding region mutations in TET genes. Despite our understanding of the biochemical activities of TET proteins, how TET loss of function promotes the onset and progression of hematopoietic malignancies is largely unknown. Here, we review recent advances in our understanding of the role of TET enzymes in lymphoid and myeloid neoplasms and highlight the importance of metabolic alterations that decrease TET activity in cancer initiation and progression.

Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Bruton's tyrosine kinase (BTK) inhibitors in treating cancer: a patent review (2010-2018).

INTRODUCTION: Bruton's tyrosine kinase (BTK) plays a critical role in the regulation of survival, proliferation, activation and differentiation of B-lineage cells. It participates by regulating multiple cellular signaling pathways, including B cell receptor and FcR signaling cascades. BTK is abundantly expressed and constitutively active in the pathogenesis of B cell hematological malignancies, as well as several autoimmune diseases. Therefore, BTK is considered as an attractive target for treatment of B-lineage lymphomas, leukemias, and some autoimmune diseases. Many industry and academia efforts have been made to explore small molecular BTK inhibitors. AREAS COVERED: This review aims to provide an overview of the patented BTK inhibitors for the treatment of cancer from 2010 to 2018. EXPERT OPINION: BTK inhibitors attract much interest for their therapeutic potential in the treatment of cancers and autoimmune diseases, especially for B cell hematological malignancies. In 2013, ibrutinib was approved by the FDA as the first-in-class BTK inhibitors for the treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), and now it is also undergoing clinical evaluation for other indications in either single or combined therapy. It is clear that BTK inhibitors can provide a promising clinical benefit in treating B-lineage lymphomas and leukemias.

Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms.

Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.