MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC

Clarithromycin enhances dasatinib-induced cell death in chronic myeloid leukemia cells, by inhibition of late stage autophagy

Lisa Schafranek, Tamara M. Leclercq, Deborah L. White & Timothy P. Hughe

Genomic translocation breakpoint sequences are conserved in BCR-ABL1 cell lines despite the presence of amplification

David M. Ross, Lisa Schafranek, Timothy P. Hughes, Mario Nicola, Susan Branford, Joannah Scor

Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia

Kinase inhibitors block proliferative signals in BCR-ABL1+ leukemic cells, but their capacity to induce apoptosis is poorly understood. Initial studies suggested that very brief exposure to kinase inhibitors was sufficient to induce apoptosis in chronic myeloid leukemia (CML) cells. However, flaws in this experimental model have subsequently been identified, leading to the conclusion that apoptosis only occurs with sustained low-level kinase inhibition. Thus, the minimum duration of complete kinase inhibition required to commit CML cells to death is unknown. Here we confirm that <1 h is insufficient to induce significant commitment to death in BCR-ABL1+ cell lines and in primary CD34+ progenitor cells, and establish that commitment to cell death only occurs if kinase inhibition is maintained for 4 h or more. Remarkably, signal transducer and activator of transcription 5 (STAT5) inhibition in combination with transient (<1 h) tyrosine kinase inhibitor (TKI) exposure proved lethal for CML progenitors, despite the reactivation of Bcr-Abl after 1 h. JAK kinase inhibition did not induce cell death in combination with transient TKI exposure. Thus, STAT5 appears to be a critical determinant of the time-dependent sensitivity of CML progenitor cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent, manner. We conclude that combining kinase inhibition with STAT5 inhibition represents a promising therapeutic approach in BCR-ABL1+ leukemias.L Schafranek, E Nievergall, J A Powell, D K Hiwase, T Leclercq, T P Hughes, and D L Whit

STAT5 is a critical component of the time-dependent sensitivity of CML cells to TKI treatment in a BCR-ABL-dependent, but JAK2-independent manner

Abstract Introduction Bcr-Abl1 is necessary and sufficient to cause chronic myeloid leukemia (CML) and as such CML cells are dependent on Bcr-Abl signalling for survival. Targeting CML cells with tyrosine kinase inhibitors (TKIs) commits cells to apoptotic cell death. Bcr-Abl constitutively activates STAT5, however the role of JAK-2 in the activation of STAT5 by Bcr-Abl is controversial. Recent studies of transient Bcr-Abl inhibition indicate that residual low levels of TKI are sufficient to maintain STAT5 inhibition in the absence of sustained Bcr-Abl inhibition. Therefore STAT5 is a highly sensitive measure of kinase activity. We hypothesized that sustained blockade of STAT5 is essential for the commitment of CML cells to apoptosis following inhibition of Bcr-Abl by TKIs. Aim To determine the role of STAT5 and JAK inhibition in the commitment of CML cells to apoptosis. Methods Factors required for CML cell death were examined in the setting of transient inhibition of Bcr-Abl by TKIs. Induction of apoptosis was assessed by Annexin V/7AAD and the clonogenic potential of CML progenitors assessed by CFU-GM assay. Bcr-Abl and apoptotic signaling pathways were interrogated by western blotting and flow cytometry. Dasatinib was used at 100 nM for potent inhibition of Bcr-Abl. Short term refers to 30 min exposure. Standard washout refers to 3 consecutive washes following potent TKI treatment. Optimal washout refers to 3 washes with 1 h equilibration at 37°C in drug free media between washes. Results In BCR-ABL+ cell lines short term, potent dasatinib exposure followed by optimal washout resulted in reactivation of Bcr-Abl and STAT5, inhibition of apoptosis (83% viable, n=3) and maintenance of colony formation in CML progenitors (CFU-GM: 85% of untreated n=3). Plasma concentrations of dasatinib vary between patients, however peak plasma levels occur up to 6 h after dosing and dasatinib remains available for up to 24 h. CML cell lines and CP-CML CD34+ progenitors were exposed to 100 nM dasatinib for 0.5-8 h before optimal washout. Cell death was achieved if TKI exposure by at least 4 h, with maximal cell death (15% viable, n=3, p=0.008) and reduction of colonies (30.1% of control, p=0.002) achieved after 8 h exposure. Comparison of 30 min and 8 h exposures to 100 nM dasatinib followed by optimal washout was performed to assess the critical signalling components required to induce apoptosis. Reactivation of Bcr-Abl, STAT5 and Erk occurred upon washout following both the 30 min and 8 h exposures, however the 8 h exposure resulted in the inhibition of STAT5 and loss of expression of STAT5 targets Mcl-1 and Bcl-xl, but not Bcl-2. In CP-CML CD34+ cells, prolonged inhibition of STAT5 was observed after 4 h exposure, following optimal washout, highlighting loss of STAT5 activity as potentially critical to irreversible induction of cell death. Continuous inhibition of STAT5 alone with pimozide (Pz) or the specific inhibitor N’-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide (herein referred to as STAT5i) led to minimal apoptosis (73% and 75% viable, respectively, n=3) when used alone. However, when combined with 30 min exposure to dasatinib (100 nM) STAT5 inhibition proved lethal in a proportion of cells despite optimal washout (57% viable +Pz and 59% +STAT5i). The clonogenic potential CML progenitors was also significantly reduced (12%, p=0.002 and 18% CFU, p=0.003) (Figure 1). The JAK1/2 kinase inhibitor ruxolitinib was used to assess the involvement of JAK1/2 in Bcr-Abl-dependent activation of STAT5. Similar to the observations with STAT5 inhibition, ruxolitinib had minimal effect on cell death as a sole agent (74% viable). However, in contrast to our observations with STAT5 inhibition, the addition of ruxolitinib to 30 min 100 nM dasatinib exposure did not induce additional cell death (70% viable, p=0.41, n=3). Conclusion STAT5 is a critical component of the time-dependent sensitivity of CML cells to TKI treatment in a Bcr-Abl-dependent, but JAK-independent manner. In contrast to previous studies describing JAK2 as a promising secondary target for the enhancement of TKI treatment of CML, we demonstrate that inhibition of STAT5 in conjunction with standard TKI therapy is a promising therapeutic strategy for the treatment of CML. Disclosures: Nievergall: CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. Lisa Schafranek, Eva Nievergall, Jason A. Powell, Devendra K. Hiwase, Deborah L. White, Timothy P. Hughes, and Tamara Leclerc

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

There are three currently identified secondary resistance mechanisms observed in patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs). These are BCR–ABL kinase domain (KD) mutations, increased BCR–ABL expression, and overexpression of drug-efflux proteins (ABCB1 and ABCG2). To investigate the interplay between these three modes of resistance, three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib to 2 μM, or dasatinib to 200 nM. Eight imatinib- and two dasatinib-resistant cell lines were established. Two imatinib-resistant K562 lines both had increased BCR–ABL expression as the apparent mode of resistance. However, when a dasatinib-resistant K562 culture was generated we observed gradually increasing BCR–ABL expression which peaked prior to identification of the T315I mutation. BCR–ABL overexpression followed by mutation development was observed in a further 4/10 cell lines, each with different KD mutations. In contrast, three imatinib-resistant K562 Dox lines exhibited only a further increase in ABCB1 expression. All TKI-resistant cell lines generated had increased IC₅₀ (dose of drug required to reduce phosphorylation of the adaptor protein p-Crkl by 50%) to imatinib, dasatinib, and nilotinib, regardless of which TKI was used to induce resistance. This suggests that currently available TKIs share the same susceptibilities to drug resistance.Carine Tang, Lisa Schafranek, Dale B. Watkins, Wendy T. Parker, Sarah Moore, Jodi A. Prime, Deborah L. White & Timothy P. Hughe

On the suitability of hydrous ruthenium oxide supports to enhance intrinsic proton conductivity in DMFC anode

Hydrous ruthenium oxides have been investigated as novel support materials for fuel cell electrocatalysts for use in DMFC applications. These oxides were chosen in particular due to their potential intrinsic proton conductivity. Pt nanoparticles have been deposited onto the new support, and the resulting catalysts characterized both structurally and electrochemically. The Pt nanoparticles are sized between 3–4 nm and are highly dispersed on the support. Transmission electron micrographs show that the individual Pt nanoparticles are covered by an amorphous coating layer – probably hydrous ruthenium oxide, in good agreement with the XPS data. Electrochemical measurements on model electrodes indicate that proton conductivity of the supporting material is strongly affected by interdiffusion of methanol. Nevertheless, initial tests on membrane electrode assemblies (MEAs) showed improved performance, particularly with respect to internal resistance, when compared to Pt-Ru black. The catalyst showed very high activity in CO stripping experiments performed on a full MEA, suggesting high catalyst utilization, despite the comparatively low Nafion® content used in the electrode layer

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC