Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Chromosomal translocations of the Mixed-lineage leukemia 1 (MLL1) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.

Ubiquitin-specific proteases (USPs) in leukemia: a systematic review.

BACKGROUND: Leukemia, a type of blood cell cancer, is categorized by the type of white blood cells affected (lymphocytes or myeloid cells) and disease progression (acute or chronic). In 2020, it ranked 15th among the most diagnosed cancers and 11th in cancer-related deaths globally, with 474,519 new cases and 311,594 deaths (GLOBOCAN2020). Research into leukemia's development mechanisms may lead to new treatments. Ubiquitin-specific proteases (USPs), a family of deubiquitinating enzymes, play critical roles in various biological processes, with both tumor-suppressive and oncogenic functions, though a comprehensive understanding is still needed. AIM: This systematic review aimed to provide a comprehensive review of how Ubiquitin-specific proteases are involved in pathogenesis of different types of leukemia. METHODS: We systematically searched the MEDLINE (via PubMed), Scopus, and Web of Science databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA) to identify relevant studies focusing on the role of USPs in leukemia. Data from selected articles were extracted, synthesized, and organized to present a coherent overview of the subject matter. RESULTS: The review highlights the crucial roles of USPs in chromosomal aberrations, cell proliferation, differentiation, apoptosis, cell cycle regulation, DNA repair, and drug resistance. USP activity significantly impacts leukemia progression, inhibition, and chemotherapy sensitivity, suggesting personalized diagnostic and therapeutic approaches. Ubiquitin-specific proteases also regulate gene expression, protein stability, complex formation, histone deubiquitination, and protein repositioning in specific leukemia cell types. CONCLUSION: The diagnostic, prognostic, and therapeutic implications associated with ubiquitin-specific proteases (USPs) hold significant promise and the potential to transform leukemia management, ultimately improving patient outcomes.

NSD3 in Cancer: Unraveling Methyltransferase-Dependent and Isoform-Specific Functions.

NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes.

L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity.

DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.

Covalent inhibition of NSD1 histone methyltransferase.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

HDAC3 functions as a positive regulator in Notch signal transduction.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.

Mannose and phosphomannose isomerase regulate energy metabolism under glucose starvation in leukemia.

Diverse metabolic changes are induced by various driver oncogenes during the onset and progression of leukemia. By upregulating glycolysis, cancer cells acquire a proliferative advantage over normal hematopoietic cells; in addition, these changes in energy metabolism contribute to anticancer drug resistance. Because leukemia cells proliferate by consuming glucose as an energy source, an alternative nutrient source is essential when glucose levels in bone marrow are insufficient. We profiled sugar metabolism in leukemia cells and found that mannose is an energy source for glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. Leukemia cells express high levels of phosphomannose isomerase (PMI), which mobilizes mannose to glycolysis; consequently, even mannose in the blood can be used as an energy source for glycolysis. Conversely, suppression of PMI expression or a mannose load exceeding the processing capacity of PMI inhibited transcription of genes related to mitochondrial metabolism and the TCA cycle, therefore suppressing the growth of leukemia cells. High PMI expression was also a poor prognostic factor for acute myeloid leukemia. Our findings reveal a new mechanism for glucose starvation resistance in leukemia. Furthermore, the combination of PMI suppression and mannose loading has potential as a novel treatment for driver oncogene-independent leukemia.

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.

Activation of ATM kinase by ROS generated during ionophore-induced mitophagy in human T and B cell malignancies.

Ataxia telangiectasia mutated (ATM), a critical DNA damage sensor, also possesses non-nuclear functions owing to its presence in extra-nuclear compartments, including peroxisomes, lysosomes, and mitochondria. ATM is frequently altered in several human cancers. Recently, we and others have shown that loss of ATM is associated with defective mitochondrial autophagy (mitophagy) in ataxia-telangiectasia (A-T) fibroblasts and B-cell lymphomas. Further, we reported that ATM protein but not ATM kinase activity is required for mitophagy. However, the mechanism of ATM kinase activation during ionophore-induced mitophagy is unknown. In the work reported here, using several ionophores in A-T and multiple T-cell and B-cell lymphoma cell lines, we show that ionophore-induced mitophagy triggers oxidative stress-induced ATMSer1981 phosphorylation through ROS activation, which is different from neocarzinostatin-induced activation of ATMSer1981, Smc1Ser966, and Kap1Ser824. We used A-T cells overexpressed with WT or S1981A (auto-phosphorylation dead) ATM plasmids and show that ATM is activated by ROS-induced oxidative stress emanating from ionophore-induced mitochondrial damage and mitophagy. The antioxidants N-acetylcysteine and glutathione significantly inhibited ROS production and ATMSer1981 phosphorylation but failed to inhibit mitophagy as determined by retroviral infection with mt-mKeima construct followed by lysosomal dual-excitation ratiometric pH measurements. Our data suggest that while ATM kinase does not participate in mitophagy, it is activated via elevated ROS.

Deregulation of mitochondrial sirtuins and OGG1-2a acts as a prognostic and diagnostic biomarker in leukemia.

Purpose: The present study was planned to explore the expression variations of mitochondrial sirtuins and the mitochondrial DNA repair enzyme OGG1-2a in leukemia patients. Oxidative stress and deacetylation levels of leukemia patients were measured in the present study. Methods: A total of 200 leukemia patients along with 200 healthy controls were evaluated using quantitative PCR, 8OXOG assay and deacetylation assay. Results: Significant deregulation of SIRT3 (p < 0.0001), SIRT4 (p < 0.0001), SIRT5 (p < 0.0001), Ki-67 (p < 0.0001) and OGG1-2a (p < 0.0001) was detected in patients versus controls. Survival analysis showed that deregulation of said genes was associated with decreased survival of leukemia patients (SIRT3: p < 0.004; SIRT4: p < 0.0009; SIRT5: p < 0.0001; OGG1-2a: p < 0.03). Receiver operating characteristic curve analysis confirmed the diagnostic values of selected genes in leukemia patients. Levels of 8OXOG adducts were measured, and significantly increased 8OXOG adduct levels were observed in patients versus controls. Conclusion: These data suggest that deregulation of SIRT3, SIRT4, SIRT5 and OGG1-2a acts as a diagnostic and prognostic marker in leukemia.

Lay abstract Leukemia is a type of blood cancer that has shown an increased rate of occurrence worldwide. Studies have shown that environmental and genetic factors are involved in the increased rate of this disease. Of the genetic factors, sirtuins (SIRT3, SIRT4 and SIRT5) and OGG1-2a have not been studied in leukemia. In the present study, the authors aimed to study the genetic/epigenetic changes in these genes in leukemia patients. Results of the present study showed involvement of selected gene variations in the increased rate of leukemia, at least in the Pakistani population.

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

PTEN action in leukaemia dictated by the tissue microenvironment.

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.

Haploinsufficiency of Dnmt1 impairs leukemia stem cell function through derepression of bivalent chromatin domains.

Epigenetic mechanisms regulating leukemia stem cells (LSCs) are an attractive target for therapy of blood cancers. Here, we report that conditional knockout of the DNA methyltransferase Dnmt1 blocked development of leukemia, and haploinsufficiency of Dnmt1 was sufficient to delay progression of leukemogenesis and impair LSC self-renewal without altering normal hematopoiesis. Haploinsufficiency of Dnmt1 resulted in tumor suppressor gene derepression associated with reduced DNA methylation and bivalent chromatin marks. These results suggest that LSCs depend on not only active expression of leukemogenic programs, but also DNA methylation-mediated silencing of bivalent domains to enforce transcriptional repression.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells.

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Expression and Activity of Matrix Metalloproteinases in Leukemia.

Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and hence play a crucial role in physiological and pathologic processes. The imbalance between the expression of MMPs and their inhibitors can be effective in leukemic cell processes such as migration, angiogenesis, survival, and apoptosis, playing a key role in the progression and prognosis of leukemia. In this review, we discuss the potential involvement of MMPs and their inhibitors in the pathogenesis and progression of leukemia by examining their role in the prognosis of leukemia. Inducing leukemic cell growth, migration, invasiveness, and angiogenesis are the main roles of MMPs in leukemia progression mediated by their degradative activity. Given the important role of MMPs in leukemia progression, further clinical trials are needed to confirm the link between MMPs' expressions and leukemia prognosis. It is hoped to use MMPs as therapeutic targets to improve patients' health by recognizing the prognostic value of MMPs in leukemia and their effect on the progression of these malignancies and their response to treatment.

Targeted Polo-like Kinase Inhibition Combined With Aurora Kinase Inhibition in Pediatric Acute Leukemia Cells.

BACKGROUND: Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. MATERIALS AND METHODS: We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. RESULTS: Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. DISCUSSION: This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.