Lilian Amrein

The phosphatidylinositol-3 kinase I inhibitor BKM120 induces cell death in B-chronic lymphocytic leukemia cells in vitro

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B‐chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high‐risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal‐101 is cytotoxic in B‐CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal‐101 in malignant B‐CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B‐CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B‐CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.

Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells

SummaryThis study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC50 values of SN38 were observed at 5 and 10xa0μM of DNA-PK inhibitors. Moreover, at 1–2xa0μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC50 of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24xa0h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

Effects of the novel DNA dependent protein kinase inhibitor, IC486241, on the DNA damage response to doxorubicin and cisplatin in breast cancer cells

SummaryThe purpose of this study was to determine the degree to which the novel DNA-PKcs inhibitor, IC486241 (ICC), synergizes the cytotoxicity of DNA damaging agents in 3 genetically diverse breast cancer cell lines. The sulforhodamine B (SRB) assay was employed as a primary screening method to determine the in-vitro cytotoxicity and the degree of synergy of ICC in combination with the topoisomerase II inhibitor, doxorubicin, or the DNA cross linking agent, cisplatin. Molecular mechanisms underlying drug toxicity were probed using immunostaining and flow cytometry, as well as, the alkaline comet assay to detect DNA damage. In this study, improved cytotoxicity and significant synergy were observed with both anticancer agents in the presence of nontoxic concentrations of ICC. Moreover, ICC decreased doxorubicin-induced DNA-PKcs autophosphorylation on Ser2056 and increased doxorubicin-induced DNA fragmentation. In conclusion, the novel DNA-PKcs inhibitor, ICC, synergistically sensitized 3 breast cancer cell lines to doxorubicin and cisplatin. Enhanced efficacy of doxorubicin was achieved by inhibiting non-homologous end joining resulting in increased accumulation of DNA damage.

Dual inhibition of the homologous recombinational repair and the nonhomologous end-joining repair pathways in chronic lymphocytic leukemia therapy.

Resistance to chlorambucil in chronic lymphocytic leukemia (CLL) has been associated with increased DNA repair. Specifically, inhibition of either c-abl, which modulates Rad51 directed homologous recombination or DNA-PK dependent nonhomologous end joining has been shown to sensitize primary CLL lymphocytes to chlorambucil. Here we report that inhibition of c-abl can result in a compensatory increase in DNA-PK and thus inhibition of both c-abl and DNA-PK optimally sensitizes CLL lymphocytes to chlorambucil. In this paper we report a drug-induced compensatory change between two DNA repair pathways with potential therapeutic implications in CLL therapy.

Primary del 17 chronic lymphocytic leukaemia lymphocytes are hypersensitive to dasatinib in vitro

Chronic lymphocytic leukaemia (CLL) is characterized by the accumulation of mature quiescent B-lymphocytes in the G0/G1 phase of the cell cycle. B-lymphocyte accumulation is likely to be a consequence of an undefined defect in the apoptotic machinery rather than an increased proliferation of leukaemic cells (Hamblin & Oscier, 1997). The prolonged survival of CLL lymphocytes has also been linked to deregulated expression and/or activity of related non receptor tyrosine kinases including members of the SRC family kinases (SFK) and ABL1. Inhibition of either c-abl (Aloyz et al, 2004) or SFK (Contri et al, 2005) results in CLL lymphocyte death in vitro. Previous results from our laboratory suggest that dasatinib cytotoxicity in CLL lymphocytes is associated with c-abl rather than SFK inhibition (Amrein et al, 2008a). Recent results of phase I–II clinical trials using dasatinib in CLL suggest that the drug might be beneficial in only a small subset (≤10%) of previously treated patients (Amrein et al, 2008b). In agreement with these clinical results, we have recently reported that dasatinib is cytotoxic to primary CLL lymphocytes in vitro but mainly at clinically unobtainable concentrations (Amrein et al, 2008a). Although dasatinib resistance was associated with the basal expression of c-abl, our study suggested that wild type TP53 is important in CLL lymphocyte homeostasis and/or survival in the presence of dasatinib (Amrein et al, 2008a). To test this hypothesis we assessed: (i) dasatinib cytotoxicity in p53 proficient (wild type) CLL lymphocytes treated with dasatinib in the presence or absence of pifithrin-α, a small molecule inhibitor of p53 transcriptional activity (Steele et al, 2008) and 2) dasatinib cytotoxicity in primary CLL lymphocytes with impaired TP53 signalling from patients diagnosed with del 17p13.1. In the eleven samples we verified the functionality of p53 signalling as described before by examining changes in p53 and its downstream target p21Cip1 (p21) after treatment with chlorambucil (Willmore et al, 2008). Briefly, the lymphocytes were treated with equivalent cytotoxic concentrations of chlorambucil (50% inhibitory concentrations [IC50’s]) for 24 h and induced p53 and p21 protein levels were monitored by Western blotting. As expected p53 and p21 protein levels were induced by chlorambucil only in TP53 wild type CLL lymphocytes (data not shown). n nAs previously reported, dasatinib IC50s in vitro in CLL lymphocytes expressing wild type TP53 were not in the clinically attainable range (mean value of 30 μmol/l, Table I) (Amrein et al, 2008a). In contrast, the dasatinib IC50s in del 17p13.1 lymphocytes were significantly lower (up to 100 times) than in the wild type TP53 lymphocytes. Moreover, in agreement with previous reports demonstrating that del 17p13.1 is associated with chemoresistance in CLL lymphocytes, we found that del 17p13.1 lymphocytes were significantly more resistant to chlorambucil that TP53 wild type CLL lymphocytes [Table I, Fig 1A (Zenz et al, 2008)]. n n n nTable I n nClinical characteristic of the patients and IC50 concentrations of dasatinib and chlorambucil in primary CLL-lymphocytes in vitro. n n n n n nFig 1 n nDasatinib decreases p53 basal expression levels in primary CLL lymphocytes expressing wild type p53. Dasatinib and chlorambucil IC50s were significantly different between CLL lymphocytes expressing wild type TP53 or del 17 (*P = 0.012). The bars represent ... n n n nIn available wild type TP53 CLL samples, pifithrin-α sensitized the lymphocytes of 2 out 3 patients (1·3 and 12-fold) (Table I). Importantly, pifithrin-α was not toxic to CLL lymphocytes when used alone. Treatment with dasatinib for twenty four hours resulted in a dose dependent reduction of p53 and p21 basal expression levels in the lymphocytes of patients expressing wild type TP53 (Fig 1B, C). In contrast, p53 levels were not affected in del 17 lymphocytes. Importantly, p21 was not detected in del 17p13.1 lymphocytes suggesting that TP53 is not functional (Fig 1D). These results are in agreement with previous reports suggesting that in the majority of CLL patients with malignant lymphocytes displaying del 17p13.1, the remaining TP53 allele is mutated (Zenz et al, 2008). n nIn addition, we found that dasatinib resistance (higher IC50) in CLL lymphocytes expressing wild type TP53 correlated with residual p53 protein levels (in respect to control) after dasatinib treatment (r = 0·8, P = 0·02, Fig 1E). As ATM is a key regulator of p53 functionality, we assessed del 11q22-23 status in del 17p13.1 lymphocytes (Table I) (Pettitt et al, 2001). Two of the three del 17p13.1 samples tested were positive for del 11q22–23. Although del 17p13.1 lymphocytes were hypersensitive when compared to p53 proficient lymphocytes, we did not find a correlation between the percentage of del 17p13.1 or del 11q22–23 in the lymphocytes and the IC50 of dasatinib. Studies regarding the role of p53 signalling (and its regulators e.g. ATM) in dasatinib sensitivity in a larger cohort of CLL samples should be informative. n nTaken together, our results suggest that p53 is important to maintain CLL lymphocyte homeostasis following exposure to dasatinib and suggest that dasatinib may be effective to treat del 17p13.1 CLL patients. The recent report of an excellent clinical response to dasatinib of a CLL patient with lymphocytes displaying del 17p13.1 supports this hypothesis (Pitini et al, 2009).

Simultaneous inhibition of ATR and PARP sensitizes colon cancer cell lines to irinotecan

Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4–3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5–27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2 −M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer.

PARP3 inhibitors ME0328 and olaparib potentiate vinorelbine sensitization in breast cancer cell lines

PurposePARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1.MethodsThe sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V, a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity.ResultsPARP3 inhibitors sensitize breast cancer cells to vinorelbine, a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold, respectively, potentiating vinorelbine-induced arrest at the G2/M boundary. In addition, PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore, olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis.ConclusionOur results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases.

Abstract 268: PARP3 inhibitors in cancer therapy

Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80. In order to examine the role of PARP3 vis-a-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2. Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC 50 ) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC 50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC 50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies. Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives: 1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples. 2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase). 3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line. The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. PARP3 inhibitors in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 268.

Abstract 1881: New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnB-cell chronic lymphocytic leukemia (CLL) is characterized by actively dividing B-lymphocytes in the lymph nodes and bone marrow, associated with the accumulation of quiescent lymphocytes in the peripheral blood of affected patients. Current treatments for this disease include chemotherapeutic (chlorambucil (CLB), cyclophosphamide, fludarabine) and immunotherapeutic agents (Rituximab, Alemtuzumab) or the combination of immunotherapy with chemotherapeutics drugs. In 2008, the FDA approved bendamustine, a bifunctional agent with a nitrogen mustard moiety and a purine analog, for the treatment of patients with CLL. Signals from the bone marrow and lymph node microenvironments protect CLL lymphocytes from drug-induced apoptosis. During treatment the enzyme-mediated repair of DNA damage can induce resistance to chemotherapeutic drugs. We have previously shown that small molecule inhibitors of c-abl (a key protein of the homologous recombinational repair pathway) such as imatinib, nilotinib or dasatinib, sensitize primary CLL lymphocytes to CLB. We also demonstrated that ZRF4, a combi-molecule designed to target c-abl (through an imatinib moiety) and to induce DNA damage (through a nitrogen mustard moiety), has a more potent cytotoxic effect than the combination of CLB plus imatinib in CLL lymphocytes. Here we report the anticancer effect of three different combi-molecules composed of a chlorambucil moiety and a dasatinib (a dual Src/c-abl inhibitor) moiety compare to the individual components in primary CLL lymphocytes in-vitro. Using MTT assays on CLL lymphocytes from 40 CLL patients, we found that the IC50 (concentration which kills 50% of the cells) of AL748, AL758 and AL816 are significantly lower than the IC50 of CLB when used alone and the combination of CLB with 0.1 μM dasatinib (median value = 1.7 μM, 0.6 μM, 0.9 μM, 11 μM and 3.1 μM respectively). Moreover, our biochemical results suggest that the mechanism of action of AL758, AL816, CLB and CLB plus dasatinib share common downstream targets including inhibition of Src kinase, chemotaxis, and cross-talk with the microenvironment along with induction of DNA damage (p53, p21, comet assay) and apoptosis (AnnexinV, cleaved caspase-3). We also determined, by the MTT assay, the cytotoxic effect of the combi-molecule AL887 composed of a bendamustine moiety and a dasatinib moiety, compare to the individual components in vitro in primary B-lymphocytes from 20 CLL patients. Our results demonstrated that this combi-molecule has a better anticancer activity than bendamustine in combination with 0.1 μM dasatinib (p<0.0005).nnDose-limiting toxicity and drug pharmacokinetics are important and limiting factors to take into account for the development of chemotherapy and will be tested in a CLL mouse model. Our results suggest that the combi-molecules AL758, AL816, and AL887 may be useful alternatives for treatment of CLL patients.nnCitation Format: Lilian Amrein, Anne-Laure Larocque, David Davidson, Lisa Peyrard, Daniel Borrelli, Bertrand Jean-Claude, Lawrence Panasci. New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2014-1881

Abstract 65: Metformin inhibits in vivo growth of MC38 colon carcinoma in the absence of LKB1 expression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnThere is evidence that metformin has anti-neoplastic activity in diabetic cancer patients. In hepatocytes, metformin administration leads to the LKB1-dependent activation of AMPK and inhibition of gluconeogenesis which can lower insulin levels. In neoplastic cells in vitro, metformin-induced activation of AMPK leads to inhibition of both protein synthesis and cell proliferation. Our previous results showed that metformin inhibits LLC1 tumor growth in mice on a high energy diet that induced hyperinsulinemia, while having no effect on tumor growth of mice on a control diet, thus raising questions regarding the roles of direct AMPK mediated anti-neoplastic effects of metformin vs indirect anti-neoplastic effects attributable to reduction of insulin levels.nnWe extended this work using MC38 colon carcinoma with shRNA to knockdown LKB1 (LKB1-). As expected, knockdown of LKB1 conferred resistance to the in vitro growth inhibitory actions of metformin. We proceeded with an in vivo study to compare growth of MC38-LKB1- and MC38 control tumors in mice on either a high energy or control diet, with or without metformin. Metformin was administered daily and the experiment was carried out with each mouse bearing a MC38-LKB1- tumor on one flank and a MC38 control tumor on the other. Metformin had no effect on the insulin levels of mice on the control diet, but significantly reduced the insulin levels of mice on the high energy diet. Tumors of mice on the high energy diet were twice the volume of tumors of mice on the control diet, regardless of LKB1 status. Metformin significantly inhibited growth of both MC38-LKB1- and MC38 control tumors in mice on the high energy diet. These observations suggest that despite its direct in vitro growth inhibitory activity involving activation of AMPK, the anti-neoplastic activity of the drug in vivo, in the context of hyperinsulinemia, is attributable to the actions of the drug on the liver. Consistent with this conclusion, we observed that metformin administration reduced insulin receptor activation in both MC38-LKB1- and MC38 control tumors of mice on the high energy diet.nnIn addition, metformin attenuated tumor growth in MC38-LKB1- cells in mice on the control diet, but had no effect on MC38 control cells. Immunoblotting confirmed that unlike MC38 control cells, LKB1- cells did not undergo autophagy in the presence of metformin. We confirmed these results in vitro and found that MC38-LKB1- treated with metformin, in conditions of low glucose, underwent apoptosis whereas LKB1- cells treated with metformin in conditions of high glucose were insensitive to metformin. MC38 control cells were equally sensitive to the growth inhibitory effects of metformin at both high and low glucose conditions. These results suggest that metformin and similar compounds deserve clinical evaluation, but that their activity may be restricted to subsets based on molecular pathology of the tumor and metabolic status of the host.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 65.