No abstract

Purpose: Thalidomide and its analogues have shown promise in the treatment of multiple myeloma but their therapeutic potential has not been evaluated in models of acute lymphoblastic leukemia (ALL). Experimental Design: We assessed the effects of the thalidomide analogue, CC-4047, on the growth and apoptosis signaling of human B cell precursor (BCP) ALL cell lines and freshly obtained childhood BCP-ALL cells grown with or without stromal cells. In addition, we studied the effects of CC-4047 on the progression and dissemination of xenotransplanted human BCP-ALL cells in nonobese diabetic/severe combined immunodeficiency mice. Results: CC-4047 reduced the proliferation of human BCP-ALL cell lines in vitro. In contrast with the antileukemic effect of cytarabin, this was more pronounced when cell lines or freshly obtained childhood BCP-ALL cells were cocultured with stromal cells. CC-4047 induced the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase in stroma-cocultured BCP-ALL cells.

Purpose: Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients.Experimental Design: Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation.Results: We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-kB pathways. We observed an activation of NF-kB after bortezomib treatment and the induction of apoptosis-related NFkB target genes such as TNFaRs after concomitant treatment, indicating a possible involvement of NF-kB as proapoptotic mediator. In this context, significantly lower NF-kB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy.Conclusion: These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols.

No abstract

The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches. Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control. From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.

A recent report from our group described that the (serotonin receptor-3)-antagonist ondansetron exhibits antiproliferative effects in the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell line REH. Furthermore, after each application of ondansetron to cultured REH cells, significant increases (+23%) in the concentration of nitric oxides (NO) were observed in the cell supernatants after 72 hours incubation in standard conditions, and this effect was found to correlate with the described antiproliferative activity. This feature was further confirmed by using mRNA dot blot hybridizations with a specific gene probe for the inducible NO-synthase (iNOS), yielding significant increases (+100%) of iNOS mRNA, which were found to widely correlate with the detected increases of NO release, and also with the previously described antiproliferative effects. The presented results are the first report on high specific pro-inflammatory features of a (serotonin receptor 3)-antagonist in a BCP-ALL cell line, which are associated with previously described antiproliferative properties.

The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B‐cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12–CXCR4 as the leading signaling axis for B‐ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient‐derived xenograft (PDX)‐ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX‐ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B‐ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2‐like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4‐mediated functions using an anti‐CXCR4 antibody treatment completely abolished testicular infiltration of PDX‐ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B‐ALL. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

<div>Abstract<p><b>Purpose:</b> Relapse of disease and subsequent resistance to established therapies remains a major challenge in the treatment of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). New therapeutic options, such as proteasome and histone deacetylase inhibitors (HDACi) with a toxicity profile differing from that of conventional cytotoxic agents, are needed for these extensively pretreated patients.</p><p><b>Experimental Design:</b> Antiproliferative and proapoptotic effects of combined HDACi/proteasome inhibitor treatments were analyzed using BCP-ALL monocultures, cocultures with primary mesenchymal stroma cells from patients with ALL, and xenograft mouse models. The underlying molecular mechanisms associated with combined treatment were determined by gene expression profiling and protein validation.</p><p><b>Results:</b> We identified the proteasome inhibitor bortezomib as a promising combination partner for HDACi due to the substantial synergistic antileukemic activity in BCP-ALL cells after concomitant application. This effect was maintained or even increased in the presence of chemotherapeutic agents. The synergistic effect of combined HDACi/BTZ treatment was associated with the regulation of genes involved in cell cycle, JUN/MAPK, PI3K/AKT, p53, ubiquitin/proteasome, and NF-κB pathways. We observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as proapoptotic mediator. In this context, significantly lower NF-κB subunits gene expression was detected in leukemia cells from patients who developed a relapse during frontline chemotherapy, compared with those who relapsed after cessation of frontline therapy.</p><p><b>Conclusion:</b> These results provide a rationale for the integration of HDACi/BTZ combinations into current childhood BCP-ALL treatment protocols. <i>Clin Cancer Res; 19(6); 1445–57. ©2013 AACR</i>.</p></div>