p185(BCR/ABL) has a lower sensitivity than p210(BCR/ABL) to the allosteric inhibitor GNF-2 in Philadelphia chromosome-positive acute lymphatic leukemia

Background: The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins are encoded as a result of the translocation, depending on whether a "minor" or "major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL) exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia. Design and methods: We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their resistance mutants. Results: The inhibitory effects of GNF-2 differed constantly between p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring resistance mutations. Conclusions: Our data provide the first evidence of a differential response of p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia

Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors

Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism

ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells

Testing breast cancer serum biomarkers for early detection and prognosis in pre-diagnosis samples

This research was funded by the National Institute for Health Research (NIHR) University College London Hospitals (UCLH) Biomedical Research Centre. UKCTOCS was core funded by the Medical Research Council, Cancer Research UK, and the Department of Health with additional support from the Eve Appeal, Special Trustees of Bart’s and the London, and Special Trustees of UCLH. OB and JFT also received support from the Eve Appeal Gynaecological Cancer Research Trust and Cancer Research UK PRC Programme Grant A12677

Single-molecule imaging of aquaporin-4 array dynamics in astrocytes

Funding: JAV’s lab is supported by grants from the European Research Council (#804581), Alzheimer’s Research UK (ARUK-PPG2019A-005), The Wellcome Trust Institutional Strategic Support Fund (204821/Z/16/Z) and RS Macdonald Charitable Trust. N. P. acknowledges funding from Medical Research Scotland [PHD-50193-2020].Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in regulating cerebrospinal fluid (CSF) circulation and facilitating the clearance of solutes from the extracellular space of the brain. Adrenergic signaling has been shown to modulate the volume of the extracellular space of the brain via AQP4 localized at the end-feet of astrocytes, but the mechanisms by which AQP4 regulates CSF inflow and outflow in the brain remain elusive. Using advanced imaging techniques, including super-resolution microscopy and single-molecule tracking, we investigated the hypothesis that β-adrenergic receptor activation induces cellular changes that regulate AQP4 array size and mobility, thus influencing water transport in the brain. We report that the β-adrenergic agonist, isoproterenol hydrochloride, decreases AQP4 array size and enhances its membrane mobility, while hyperosmotic conditions induce the formation of larger, less mobile arrays. These findings reveal that AQP4 arrays are dynamic structures, responsive to adrenergic signals and osmotic changes, highlighting a novel regulatory mechanism of water transport in the brain. Our results provide insights into the molecular control of CSF circulation and extracellular brain space volume, laying the groundwork for understanding the relationship between astrocyte water transport, sleep physiology, and neurodegeneration.Peer reviewe

The functional interplay between the t(9;22)-associated fusion proteins BCR/ABL and ABL/BCR in Philadelphia chromosome-positive Acute Lymphatic Leukemia

The hallmark of Philadelphia chromosome positive (Ph+) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL

The leukemogenic potential of t(9;22) fusion proteins.

<p>(A) Schematic representation of the experimental procedure. Sca1⁺ bone marrow cells were infected with the indicated retroviruses and inoculated into sub-lethally irradiated mice. Empty vector-transduced cells were used as control; (B) Kaplan Maier curves present the probability of survival upon primary induction of leukemia and re-transplantation of leukemic cells in order to induce secondary (II) leukemia; (C) May-Grünwald-Giemsa staining of cytospins from BM and spleens of p185^{<i>BCR/ABL</i>} and p96+p185-positive leukemia of one representative mouse in each group; (D) Expression of differentiation specific surface markers (Mac1: monocytes- macrophage, Gr1: granulocytes and B220: mature B-cells) of one representative mouse in p185^{<i>BCR/ABL</i>} and <i>p96</i>^{<i>ABL/BCR</i>} + p185^{<i>BCR/ABL</i>} (p96+p185) groups. (E) Co-expression of differentiation specific surface markers (Mac1/Gr-1—myeloid leukemia; B220/CD19: B-cell leukemia) of one representative mouse in p185^{<i>BCR/ABL</i>} and <i>p96</i>^{<i>ABL/BCR</i>} + p185^{<i>BCR/ABL</i>} (p96+p185) groups and secondary transplanted sup.</p

Targeting p96^{<i>ABL/BCR</i>} in Ph⁺ ALL cells.

<p>(A) SupB15 and K562 cells were lentivirally transduced with shRNA against p96^{<i>ABL/BCR</i>} (siR961 and siR962) and a control shRNA (NTC). The expression of p96^{<i>ABL/BCR</i>} and/or BCR was detected by immunoblotting using anti-BCR antibody. Tubulin was used as loading control. Proliferation was measured using XTT-assay after 3 days. One representative experiment in triplicates ± SD of at least three yielding similar results is given; (B) The effect of targeting p96^{<i>ABL/BCR</i>} by shRNA in SupB15 on STAT5 and ERK1/2 pathway was detected using the indicated antibodies; (C) Down-regulation of p96^{<i>ABL/BCR</i>} in Ph⁺ ALL PD-LTCs by shRNA. Ph⁺ ALL PD-LTCs—PH: fully TKI-responsive; BV: TKI-resistant; as controls were used: HP (Ph^- ALL patient) and VG: t(12;9)-TEL/ABL-positive ALL. The effect of shRNAs on the expression of p96^{<i>ABL/BCR</i>} was tested by immunoblotting using the indicated antibodies and by q-RT-PCR for PH and BV. The Ct values were normalized to that of GAPDH and results are represented as 2^-ΔΔCt. Proliferation was measured by XTT-assay. The mean of at least experiments is given ± SD.</p