Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution.

Targeting transcription-coupled nucleotide excision repair overcomes resistance in chronic lymphocytic leukemia.

Treatment resistance becomes a challenge at some point in the course of most patients with chronic lymphocytic leukemia (CLL). This applies to fludarabine-based regimens, and is also an increasing concern in the era of more targeted therapies. As cells with low-replicative activity rely on repair that triggers checkpoint-independent noncanonical pathways, we reasoned that targeting the nucleotide excision repair (NER) reaction addresses a vulnerability of CLL and might even synergize with fludarabine, which blocks the NER gap-filling step. We interrogated here especially the replication-independent transcription-coupled-NER ((TC)-NER) in prospective trial patients, primary CLL cultures, cell lines and mice. We screen selected (TC)-NER-targeting compounds as experimental (illudins) or clinically approved (trabectedin) drugs. They inflict transcription-stalling DNA lesions requiring TC-NER either for their removal (illudins) or for generation of lethal strand breaks (trabectedin). Genetically defined systems of NER deficiency confirmed their specificity. They selectively and efficiently induced cell death in CLL, irrespective of high-risk cytogenetics, IGHV status or clinical treatment history, including resistance. The substances induced ATM/p53-independent apoptosis and showed marked synergisms with fludarabine. Trabectedin additionally perturbed stromal-cell protection and showed encouraging antileukemic profiles even in aggressive and transforming murine CLL. This proof-of-principle study established (TC)-NER as a mechanism to be further exploited to resensitize CLL cells.

The regulatory interaction of EVI1 with the TCL1A oncogene impacts cell survival and clinical outcome in CLL.

Dysregulated T-cell leukemia/lymphoma-1A (TCL1A), a modulator in B-cell receptor (BCR) signaling, is causally implicated in chronic lymphocytic leukemia (CLL). However, the mechanisms of the perturbed TCL1A regulation are largely unknown. To characterize TCL1A-upstream networks, we functionally screened for TCL1A-repressive micro-RNAs (miRs) and their transcriptional regulators. We identified the novel miR-484 to target TCL1A's 3'-UTR and to be downregulated in CLL. In chromatin immunoprecipitations and reporter assays, the oncogenic transcription factor of myeloid cells, EVI1, bound and activated the miR-484 promoter. Most common in CLL was a pan-EVI1 transcript variant. EVI1 protein expression revealed distinct normal-tissue and leukemia-associated patterns of EVI1/TCL1A co-regulation. EVI1 levels were particularly low in TCL1A-high CLL or such cellular subsets. Global gene expression profiles from a 337-patient set linked EVI1 networks to BCR signaling and cell survival via TCL1A, BTK and other molecules of relevance in CLL. Enforced EVI1, as did miR-484, repressed TCL1A. Furthermore, it reduced phospho-kinase levels, impaired cell survival, mitigated BCR-induced Ca-flux and diminished the in vitro ibrutinib response. Moreover, TCL1A and EVI1 showed a strongly interactive hazard prediction in prospectively treated patients. Overall, we present regressive EVI1 as a novel regulatory signature in CLL. Through enhanced TCL1A and other EVI1-targeted hallmarks of CLL, this contributes to an aggressive cellular and clinical phenotype.

Stromal cells modulate TCL1 expression, interacting AP-1 components and TCL1-targeting micro-RNAs in chronic lymphocytic leukemia.

The tissue microenvironment in chronic lymphocytic leukemia (CLL) has an increasingly recognized role in disease progression, but the molecular mechanisms of cross talk between CLL cells and their microenvironment remain incompletely defined. Bone marrow stromal cells (BMSC) protect CLL cells from apoptosis in a contact-dependent fashion, and have been used for the identification of key pathways such as the CXCR4-CXCL12 axis. To further dissect the molecular impact of BMSC on survival and the molecular activation signature of CLL cells, we co-cultured CLL cells with different BMSC. Gene expression profiling of CLL cells revealed that the lymphoid proto-oncogene TCL1 was among the top genes upregulated in CLL cells by BMSC. TCL1 mRNA and protein upregulation by BMSC was paralleled by decreases of TCL1-interacting FOS/JUN, and confirmed by qRT-PCR, immunoblotting, immunoprecipitations, and flow cytometry. Stroma mediated increases in TCL1 were also associated with decreased levels of TCL1-regulatory micro-RNAs (miR-29b, miR-181b, miR-34b). These findings demonstrate that the microenvironment has a proactive role in the regulation of the known signaling enhancer and pro-survival molecule TCL1 in CLL. This provides a further rationale for therapeutically targeting the cross talk between CLL and BMSC.

Inhibition of human DNA topoisomerase I by new DNA minor groove ligands: derivatives of oligo-1,3-thiazolecarboxamides.

A series of novel thiazole-containing oligopeptides (oligo-1,3-thiazolecarboxamides) interesting specifically with the minor groove of DNA was shown to inhibit human DNA topoisomerase I (topo I). Inhibitory effects of thiazole-containing oligopeptides (TCO) increase with the number of thiazole units in such compounds. Inhibitory properties of TCO containing 3 or 4 thiazole units were shown to be 3-10 times better than those of the well-known natural antibiotic, distamycin A containing pyrrole rings. The structure of various additional groups attached to the N-terminus and C-terminus of TCO had no significant effect on TCO interaction with the complex of DNA and topo I. TCO were shown to be capable of binding with double-stranded DNA (dsDNA), and the majority of TCO analyzed were more effective in binding with dsDNA than distamycin A. Possible reasons for the different effects of distamycin A and TCO on the reaction of relaxation catalyzed by topo I are discussed.

Inhibition of HIV-1 integrase-catalysed reaction by new DNA minor groove ligands: the oligo-1,3-thiazolecarboxamide derivatives.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the retrovirus, responsible for catalysing the insertion of the viral genome into the host cell chromosome. For this reason it provides an attractive target for antiviral drug design. We synthesized a series of novel thiazole (Tz)-containing oligopeptides (TCOs; oligo-1,3-thiazolecarboxamides), specifically interacting within the minor groove of DNA. The oligocarboxamide derivatives contained 1-4 Tz rings and different N- and C-terminal groups. The effect of these oligocarboxamides on the HIV-1 IN-catalysed reaction was investigated. Some of the compounds were able to inhibit the reaction. The inhibitory effect of the TCOs increased with the number of Tz units. The structure of various additional positively and/or negatively charged groups attached to the N- and C-termini of TCOs had a pronounced effect on their interaction with the DNA substrate complexed to IN. Modified TCOs having a better affinity for this complex should provide a rationale for the design of drugs targeting the integration step.

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of human DNA topoisomerase I (topo I) with specific sequence oligodeoxynucleotides (ODNs) of different length and structure has been investigated. All the ODNs used were shown to be effective enzyme inhibitors and to inhibit the topo I catalyzed relaxation of scDNA in a competitive manner. Among two DNA regions (A and B) required for topo I-mediated DNA cleavage, the former was found to display the higher affinity for the enzyme. The enzyme's affinity for ODNs corresponding to the scissile strand (five and nine nucleotide units in length) is about 2-4 orders of magnitude higher than that for non-specific ODNs of the same length. Topo I can efficiently recognize even extremely short specific ODNs containing only two or three bases (AGA and pAG, Ki = 15 and 60 microM, respectively): the sequence AAGA (Ki = 10 microM) is essential for tight DNA binding to topo I. The affinities of ODNs corresponding to the non-scissile strand are significantly lower. The ligand's affinity increases with its length. Additionally, about a ten-fold enhancement of specific sequence affinity occurs due to stable duplex formation during enzyme preincubation with ligands before addition of scDNA. We believe the possibility of using the short specific oligonucleotides and its derivatives as topoisomerase I-targeting drugs could not be excluded.

High affinity interaction of mouse DNA topoisomerase I with di- and trinucleotides corresponding to specific sequences of supercoiled DNA cleaved chain.

Recently mouse DNA topoisomerase I (topo) was shown to possess high affinity for a single-stranded AAGACTTAG nonanucleotide (K(i) = 2.0 microM) corresponding to the scissile strand of the minimal DNA duplex, which is necessary for cleavage of supercoiled DNA. In order to determine the most important part of the above sequence for the DNA recognition by topo, the interactions of the enzyme with a set of extremely short (2-5 nucleotides in length) oligonucleotides corresponding to different parts of the nonanucleotide have been investigated. The affinities of different oligonucleotides corresponding to the CTTAG part of the sequence (K(i) = 0.13-0.92 mM) were shown to be significantly lower than that for the AAGA tetranucleotide (K(i) = 9.0 microM). Topo effectively recognized even short oligonucleotides containing only two or three bases (AGA and pAG, K(i) = 20 and 50 microM). We suppose that oligonucleotides having a high afffinity to the enzyme can offer a unique opportunity for the rational design of topoisomerase-targeting drugs.