Investigation of dual Src/c-Abl tyrosine kinase inhibition as a novel therapeutic approach for chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world, and remains incurable with current chemotherapy. Although CLL was long-regarded as an autonomous accumulation of functionally incompetent lymphocytes that escape apoptosis, significant rates of clonal proliferation and death have now been elegantly demonstrated in CLL patients in vivo. This, coupled with the high rates of spontaneous apoptosis observed on ex-vivo culture, confirms that CLL is a dynamic disorder, in which the tumour microenvironment is central to leukemic cell survival. Recent advances in CLL cell biology implicate signalling through the B cell antigen receptor (BCR) in the pathogenesis and progression of the disease. The absence of significant somatic hypermutation of the immunoglobulin heavy chain variable region (IgVH), which largely correlates with the expression of ZAP-70, in CLL cells is a significant adverse prognostic marker. CLL cases expressing an unmutated IgVH gene generally retain the ability to signal through the BCR. Components of the BCR signalling pathway are therefore attractive novel therapeutic targets, with potential selectivity for adverse prognostic groups. The non-receptor tyrosine kinases Lyn (a Src kinase) and c-Abl are both required for effective BCR signalling. Both are over-expressed, and constitutively active in CLL, and inhibition of either induces a degree of apoptosis. Dasatinib is a Src/c-Abl tyrosine kinase inhibitor in clinical use in chronic myeloid leukaemia. The main objective of this project was to conduct translational studies to determine the anti-leukaemic effects of dasatinib on CLL cells in vitro. While the Src kinase inhibitor PP2, and the c-Abl inhibitor imatinib induced apoptosis of CLL cells at micromolar concentrations, dasatinib induced apoptosis of CLL cells at clinically achievable nanomolar concentrations, with an EC50 in the region of 10-30 nM, and plateau in effect above 100 nM. CLL cell treatment with 100 nM dasatinib for 48 hr led to a mean reduction in viability of 33.7%, but with significant inter-sample variability. No correlation was observed between dasatinib sensitivity and the established prognostic factors clinical stage, ZAP-70 status, or cytogenetic subgroup. Notably, CLL cells known to contain the 17p deletion, resulting in p53 dysfunction, responded similarly to other samples. Apoptosis induced by dasatinib involved loss of mitochondrial membrane potential and was caspase-dependent. Although dasatinib treatment alone rarely induced apoptosis of over 50% of CLL cells, synergy was observed between dasatinib and the current first-line chemotherapeutic agents fludarabine and chlorambucil. Moreover, dasatinib exhibited synergy with a novel Bcl-2 inhibitor, and the HSP90 inhibitor 17-DMAG. Recently, antigen-independent ‘tonic’ BCR signalling has been linked to the pathogenesis of B cell lymphomas. Tonic signalling is proposed to be mediated by basal activity of Lyn and Syk kinases recruited to the BCR. As Syk is also over-expressed in CLL, we hypothesised that dasatinib sensitivity may correlate with inhibition of components of tonic BCR signal transduction. Indeed, CLL cells contained constitutively phosphorylated SykY348. Furthermore, a significant inverse correlation was observed between basal SykY348 phosphorylation and dasatinib sensitivity in individual samples, suggesting its’ utility as a biomarker of response. Dasatinib consistently inhibited an increase in SykY348 phosphorylation on BCR crosslinking. In addition, dasatinib inhibited calcium flux, and prevented Akt and MAPK phosphorylation on BCR stimulation. Moreover, dasatinib prevented up-regulation of Mcl-1 and blocked the increase in CLL cell survival observed on prolonged BCR stimulation, confirming inhibition of BCR signalling as a functionally relevant treatment strategy in CLL. Although dasatinib prevented CXCR4 down-regulation following BCR stimulation of CLL cells, dasatinib also specifically inhibited PI-3K/Akt activation upon CXCR4 stimulation by SDF-1, resulting in reduced actin polymerization and migration following SDF-1 stimulation. While the full translational implications of these observations remain to be determined, these data demonstrate that the anti-leukaemic effects of dasatinib extend beyond direct inhibition of BCR signal transduction. It is well recognised that bone marrow (BM) stromal cells and blood-derived ‘nurse-like’ cells protect CLL cells from spontaneous apoptosis in vitro. More recently, proliferating CLL cells have been identified within specialised ‘proliferation centres’, admixed with appreciable numbers of T lymphocytes, predominantly activated CD4+ T cells expressing CD40 ligand (CD154), and interleukin 4 (IL-4). CD40/IL-4 stimulation of CLL cells in vitro leads to up-regulation of the anti-apoptotic Bcl-2 family proteins Bcl-xL and Mcl-1, and the pro-proliferative protein survivin, mimicking the expression profile of CLL cells isolated from patient lymph nodes (LNs). We were interested to determine whether the effects of dasatinib on CLL cells were modulated by these microenvironmental factors. To achieve this, CLL cells were co-cultured with either the murine BM stromal cell line NT-L, or NT-L cells stably transfected to express CD154, the latter with IL-4 added to the culture medium (154L/IL-4 system). The pro-apoptotic effect of dasatinib in CLL cells was abrogated by stromal cell co-culture, with or without CD154 and IL-4. Stromal cell-mediated resistance to dasatinib involved Akt and MAPK signalling, as evidenced by the ability of both the PI-3K inhibitor LY294002, and the MEK inhibitor PD98059 to restore dasatinib sensitivity of cells in NT-L co-culture. 154L/IL-4 co-culture activated multiple MAPK, associated with up-regulation of Bcl-xL, Mcl-1, and survivin, which was not inhibited by dasatinib. Dasatinib also failed to inhibit CLL cell proliferation in the 154L/IL-4 system. While dasatinib retained the ability to sensitise CLL cells to both fludarabine and chlorambucil in NT-L co-culture, the addition of CD154 and IL-4 rendered cells resistant to all drug combinations. Dasatinib did however retain the ability to sensitise CLL cells to the HSP90 inhibitor 17-DMAG in both NT-L and 154L/IL-4 co-culture. In conclusion, these studies demonstrate that dasatinib offers much as a novel therapeutic strategy for CLL, overcoming pro-survival signalling through the BCR. However, our data suggest that dasatinib may be best utilised in combination treatment strategies with agents that can target antigen-independent signalling networks within the microenvironment. Collectively, this work provides valuable information that will inform future clinical trials of Src/c-Abl inhibitors in CLL

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKC subversion induces up-regulation of PKC II expression in B lymphocytes

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis

Inhibition of NF-κB-mediated signaling by the cyclin-dependent kinase inhibitor CR8 overcomes pro-survival stimuli to induce apoptosis in chronic lymphocytic leukemia cells

Purpose: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. Experimental Design: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry–based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. Results: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. Conclusions: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL

Dasatinib inhibits CXCR4 signaling in chronic lymphocytic leukaemia cells and impairs migration towards CXCL12

Chemokines and their ligands play a critical role in enabling chronic lymphocytic leukaemia (CLL) cells access to protective microenvironmental niches within tissues, ultimately resulting in chemoresistance and relapse: disruption of these signaling pathways has become a novel therapeutic approach in CLL. The tyrosine kinase inhibitor dasatinib inhibits migration of several cell lines from solid-organ tumours, but effects on CLL cells have not been reported. We studied the effect of clinically achievable concentrations of dasatinib on signaling induced by the chemokine CXCL12 through its' receptor CXCR4, which is highly expressed on CLL cells. Dasatinib pre-treatment inhibited Akt and ERK phosphorylation in CLL cells upon stimulation with CXCL12. Dasatinib also significantly diminished the rapid increase in actin polymerisation observed in CLL cells following CXCL12 stimulation. Moreover, the drug significantly inhibited chemotaxis in a transwell assay, and reduced the percentage of cells able to migrate beneath a CXCL12-expressing murine stromal cell line. Dasatinib also abrogated the anti-apoptotic effect of prolonged CXCL12 stimulation on cultured CLL cells. These data suggest that dasatinib, akin to other small molecule kinase inhibitors targeting the B-cell receptor signaling pathway, may redistribute CLL cells from protective tissue niches to the peripheral blood, and support the investigation of dasatinib in combination strategies

AKT/mTORC2 inhibition activates FOXO1 function in CLL cells reducing B cell receptor-mediated survival

Purpose: To determine whether inhibition of mechanistic target of rapamycin (mTOR) kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Experimental Design: Stratification of mTOR activity was carried out in primary CLL patient samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Results: Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Conclusions: Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib

Fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin with gemtuzumab ozogamicin improves event-free survival in younger patients with newly diagnosed aml and overall survival in patients with npm1 and flt3 mutations

Purpose To determine the optimal induction chemotherapy regimen for younger adults with newly diagnosed AML without known adverse risk cytogenetics. Patients and Methods One thousand thirty-three patients were randomly assigned to intensified (fludarabine, cytarabine, granulocyte colony-stimulating factor, and idarubicin [FLAG-Ida]) or standard (daunorubicin and Ara-C [DA]) induction chemotherapy, with one or two doses of gemtuzumab ozogamicin (GO). The primary end point was overall survival (OS). Results There was no difference in remission rate after two courses between FLAG-Ida + GO and DA + GO (complete remission [CR] + CR with incomplete hematologic recovery 93% v 91%) or in day 60 mortality (4.3% v 4.6%). There was no difference in OS (66% v 63%; P = .41); however, the risk of relapse was lower with FLAG-Ida + GO (24% v 41%; P < .001) and 3-year event-free survival was higher (57% v 45%; P < .001). In patients with an NPM1 mutation (30%), 3-year OS was significantly higher with FLAG-Ida + GO (82% v 64%; P = .005). NPM1 measurable residual disease (MRD) clearance was also greater, with 88% versus 77% becoming MRD-negative in peripheral blood after cycle 2 (P = .02). Three-year OS was also higher in patients with a FLT3 mutation (64% v 54%; P = .047). Fewer transplants were performed in patients receiving FLAG-Ida + GO (238 v 278; P = .02). There was no difference in outcome according to the number of GO doses, although NPM1 MRD clearance was higher with two doses in the DA arm. Patients with core binding factor AML treated with DA and one dose of GO had a 3-year OS of 96% with no survival benefit from FLAG-Ida + GO. Conclusion Overall, FLAG-Ida + GO significantly reduced relapse without improving OS. However, exploratory analyses show that patients with NPM1 and FLT3 mutations had substantial improvements in OS. By contrast, in patients with core binding factor AML, outcomes were excellent with DA + GO with no FLAG-Ida benefit

Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer

Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK

Clinical sample details.

<p>ND = not determined</p

Dasatinib does not inhibit proliferation of CLL cells induced by CD154 and IL-4.

<p><b>A:</b> The panels show the assessment of cell division by successive dilution of CFSE at days 6, 9, and 12 of co-culture in the CD154/IL-4 system in one representative experiment. <b>B:</b> The graph shows the mean (±SEM) percentage of cells in each cell division at day 12 from six independent experiments (samples 34, 41, 46, 68, 69 and 70). <b>C:</b> The graph shows the mean (±SEM) cell counts determined for each condition and time point in all six experiments.</p

Dasatinib inhibits actin polymerisation and migration of CLL cells in response to CXCL12.

<p><b>A:</b> Analysis of the effect of dasatinib on actin polymerization following CXCL12 treatment of CLL cells. Results are expressed relative to the untreated control prior to CXCL12 stimulation. The graph shows the mean (±SEM) values for five independent experiments using different CLL samples (samples 7, 41, 45, 46 and 62). * indicates p<0.05. <b>B:</b> The graph shows the effect of increasing concentrations of dasatinib on chemotaxis of CLL cells towards CXCL12. Data are expressed as the average number (±SEM) of migrated cells from five independent experiments (samples 21, 35, 45, 46 and 62). <b>C:</b> The graph shows the percentage of CLL cells able to migrate beneath a stromal cell layer (pseudoemperipolesis) in the presence or absence of dasatinib. Results are expressed as the mean (±SEM) percentage of migrated cells in each condition (n = 8; samples 12, 18, 32, 46, 52, 64, 68 and 69). <b>D:</b> CLL cells (1×10⁶) were incubated in conditions as shown for 48 hr, then apoptosis was assessed by Annexin/Viaprobe staining. The graph shows the mean (±SEM) viabilities of cells, relative to the viability of untreated control cells (n = 6; samples 12, 36, 41, 44, 45 and 62).</p