Kerstin M. Kampa

Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage–response thresholds

The expression of ASPP2 (53BP2L), a proapoptotic member of a family of p53-binding proteins, is frequently suppressed in many human cancers. Accumulating evidence suggests that ASPP2 inhibits tumor growth; however, the mechanisms by which ASPP2 suppresses tumor formation remain to be clarified. To study this, we targeted the ASPP2 allele in a mouse by replacing exons 10–17 with a neoR gene. ASPP2−/− mice were not viable because of an early embryonic lethal event. Although ASPP2+/− mice appeared developmentally normal, they displayed an increased incidence of a variety of spontaneous tumors as they aged. Moreover, γ-irradiated 6-week-old ASPP2+/− mice developed an increased incidence of high-grade T cell lymphomas of thymic origin compared with ASPP2+/+ mice. Primary thymocytes derived from ASPP2+/− mice exhibited an attenuated apoptotic response to γ-irradiation compared with ASPP2+/+ thymocytes. Additionally, ASPP2+/− primary mouse embryonic fibroblasts demonstrated a defective G0/G1 cell cycle checkpoint after γ-irradiation. Our results demonstrate that ASPP2 is a haploinsufficient tumor suppressor and, importantly, open new avenues for investigation into the mechanisms by which disruption of ASPP2 pathways could play a role in tumorigenesis and response to therapy.

New insights into the expanding complexity of the tumor suppressor ASPP2

Apoptosis Stimulating Protein of p53-2, ASPP2, aka 53BP2L, (encoded by TP53BP2) is a pro-apoptotic member of a family of p53 binding proteins. ASPP2 expression is frequently suppressed in human cancers and numerous studies have consistently demonstrated that ASPP2 inhibits cell growth as well as stimulates apoptosis⎯at least in part through a p53-mediated pathway. Two independent mouse models have shown that ASPP2 is a haplo-insufficient tumor suppressor and underscore the importance of the role of ASPP2 in human cancer. However, mounting evidence suggests that the mechanism(s) of action for ASPP2 are complex and likely extend beyond stimulation of apoptotic programs. Data highlighting this expanding spectrum of potential ASPP2-mediated pathways is summarized along with new results from recent in vivo models suggesting new avenues for investigation.

The FLT3 inhibitor tandutinib (formerly MLN518) has sequence-independent synergistic effects with cytarabine and daunorubicin

AML remains a difficult disease to treat. Despite response to induction chemotherapy, most patients ultimately relapse. Further, among elderly patients, aggressive therapy options are often limited due to other medical conditions and decreased tolerance of these patients to conventional chemotherapy. Internal tandem duplications (ITD) of the FLT3 juxtamembrane domain occur in 20-30% of AML patients and predict poor outcome. First clinical data with the FLT3 inhibitor tandutinib demonstrated antileukemic activity in approximately half of the patients - predominantly with FLT3 ITD-positive AML. But the data also show that optimal use of tandutinib will require combination therapy with cytotoxic agents. Notably, single agent tandutinib has not been associated with myelosuppression, mucositis or cardiac toxicity - the dose limiting toxicities of AML chemotherapy. We determined the feasibility of combining tandutinib with the standard “3+7” induction regimen in AML and show that, in contrast to other structurally unrelated FLT3 inhibitors recently evaluated in clinical trials, the use of tandutinib displayed application sequence independent synergistic antileukemic effects in combination with cytarabine and daunorubicin. Strong synergistic antiproliferative and proapoptotic effects were thereby predominantly seen on FLT3 ITD-positive blasts. Further we demonstrate, that addition of tandutinib may allow dose reduction of chemotherapy without loss of overall antileukemic activity – but with a resultant decrease of potential side effects. This approach might be an interesting novel strategy especially in the treatment of elderly/comorbid patients. Our data provide a rationale for combining tandutinib with induction chemotherapy in intensified as well as in dose reduction protocols for FLT3 ITD-positive AML.

Abstract 2238: Role of phosphoinositide-dependent protein kinase-1 (PDK1) in leukemogenesis of acute leukemia

The Phosphoinositide-dependent Protein Kinase-1 (PDK1) is a key regulator of AKT signaling by phosphorylation of a threonine residue at codon 308. PDK1 itself is activated by PI3kinases and plays an important role in growth factor signaling. We previously demonstrated that AKT is globally activated in acute leukemia, e.g. via gain-of function tyrosine kinase mutations. Consecutively we have shown that tyrosine kinase inhibitors suppress codon 473 phosphorylation - but leave Thr308 activated, hypothetically via activation by growth factors. Accordingly, we show that phosphorylation status of AKT in bone marrow leukemia blasts is higher compared to circulating blasts, which favors the hypothesis of a protective bone marrow microenvironment due to the vast occurrence of growth factors and chemokines, which activate PI3K/AKT signaling. We aimed to investigate whether activation of AKT(Thr308) is crucial for the maintenance of leukemia cells and leukemogenesis and silenced PDK1 by retrovirally transducing different short hairpin RNA constructs cloned into a mammalian vector into AML cell lines. Proliferation capacity of the PDK1-silenced cells in comparison to an empty vector control was studied using an XTT-based assay. Cell lines with suppressed transcription of PDK1 revealed a significantly diminished proliferation capacity compared to the empty vector controls. To evaluate potency of tumor engraftment and aggressiveness of disease in vivo, we transplanted stably transfected PDK1-shRNA silenced acute leukemia cells into NOD/SCID mice. Isogenic cells transfected with an empty vector were used as the control group. Mice were regularly monitored for disease symptoms and leukemic blast count. Already on day 8, the peripheral blast count in the control mice revealed a faster increase with higher cell counts (∼ 4 x 10ex3/microL) compared to the PDK1-silenced group (∼ 0,5 x 10ex3/microL). Over time, all mice finally developed leukemia, with the control mice to be the first. In addition, we tested two novel PI3K/mTOR inhibitors, NVP-BEZ235 and NVP-BGT226, with regard to the antiproliferative capacity in ex vivo leukemia blasts. Both agents revealed potent antiproliferation with NVP-BGT226 the more potent agent. NVP-BGT226 induced apoptosis in ex vivo blasts in a subset of patients, with an IC50 in the low nanomolar range. In summary, we here identify the PDK1-AKT signal transduction pathway to be crucial for aggressiveness of disease. Of note, it was not possible to suppress development of leukemia completely, arguing for a multistep mechanism of leukemogenesis. Our data supports descriptive expression analysis of PDK1 or AKT(Thr308) that link high expression with unfavorable clinical outcome. Therefore, targeting the PI3K/AKT axis which activates PDK1 in acute leukemia may be beneficial to improve therapy outcome in a subset of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2238. doi:1538-7445.AM2012-2238

Abstract 4658: Delta9-Tetrahydrocannabinol induces apoptosis via Cannabinol Receptor 1 and modulates methylation of oncogenes and tumorsuppressors in blasts derived from patients with acute lympoblastic or myeloid leukemia

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Some cannabinol derivatives are said to possess antitumor efficacy. We have evidence that the CB1/2 cannabinoid receptor agonist Delta9-Tetrahydrocannabinol (THC), the major psychoactive component of marijuana, may have contributed to disease control in a patient with acute myeloid leukemia. We speculated that THC may induce apoptosis in native leukemic blasts and thus treated various leukemia cell lines and ex vivo blasts derived from patients with either acute lymphoblastic or myeloid leukemia in dilution series with THC. The antiproliferative effect was measured using an XTT-based assay. Induction of apoptosis was assessed by annexinV-based immunofluorescence analysis. The CB1 antagonist [LY320135][1] or JTE-907, a selective CB2 inverse ligand agonist, were used to confirm involvement of CB1 (mainly expressed in brain tissue) or CB2 (expression in hematologic/lymphoid cells). THC was obtained with permission of the Bundesopiumstelle, Germany. Indeed, we demonstrate potent antiproliferative and proapoptotic efficacy of Delta9-THC in a dose dependent manner in leukemia cell lines of lymphoid and myeloid origin. Pretreatment with [LY320135][1], but not with JTE-907, resulted in a dramatic abrogation of the antileukemic effect seen with THC monotherapy, arguing that THC-induced apoptosis is mediated via the cannabinoid receptor CB1. Besides a proapoptotic effect, THC was able to abrogate cellular differentiation blockage as observed in a change of morphology. Of note, THC was able to induce apoptosis in a subset of leukemia samples derived from patients with acute myeloid (mainly undifferentited AML) or lymphoblastic leukemia. Due to the patient cohort responsive to THC therapy, we speculated that epigenetic modifications might be associated with MLL (mixed lineage leukemia) methyltransferase function. In a global DNA methylation gene array, we could demonstrate that THC leads to modulation of methylation status of histone deacetylases, oncogenes and tumorsuppressors, some reported to be regulated by MLL. Ongoing work is focussing on validation whether THC treatment can be linked to MLL activity using an siRNA approach. In conclusion, while not being able to directly correlate the disease course of the above described patient to marijuana abuse, our in vitro results nevertheless demonstrate that THC mediates antileukemic activity in blasts of some patients ex vivo including that of our patient. Clinical evaluation of cannabinol receptor agonists as low-toxic agents could thus be considered in selected cases of patients with acute leukemia and this approach should further be followed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4658. doi:1538-7445.AM2012-4658 [1]: /lookup/external-ref?link_type=GEN&access_num=LY320135&atom=%2Fcanres%2F72%2F8_Supplement%2F4658.atom

Abstract 2496: Epothilones synergistically induce CRC cell death in combination with standard chemotherapy in part through a p53-dependent pathway

Epothilones are a novel class of microtubule stabilizing agents with known antitumor activity. However, clinical response rates to epothilones vary widely, and the underlying molecular mechanisms that predict response to therapy remain to be clarified. Loss of p53 tumor suppressor pathway function is a common event in many cancers, including colorectal cancer (CRC). However, it is yet unknown how p53 status affects the response to epothilones in CRC. To explore the role of the p53 pathway in epothilone antitumor activity, we quantified the antitumor efficacy of Patupilone (Pp) and Ixabepilone (Ix) in combination with standard CRC chemotherapy agents in isogenic HCT116 p53+/+ and p53-/- as well as in isogenic RKO and SW48 p53+/+ and -/- cell lines. Cells were treated with oxaliplatin (Ox), 5-flourouracil (5FU), irinotecan (Ir) as well as Pp and Ix in different doses and combinations, and cell cycle perturbations and apoptosis induction were determined. We found that all the tested agents induced cell death in a dose- and p53 status-dependent manner. Both epothilones displayed enhanced antitumor activity in p53+/+ cells compared to p53-/- cells, suggesting a p53 dependent mechanism; however, Pp induced apoptosis more efficiently than Ix as single agent (IC50s for Pp in p53+/+ cells 15nm and in p53-/- cells 50nm; IC50s for Ix 50nm and >100nm respectively). Intriguingly, Pb and Ix induced a G2/M checkpoint arrest in p53+/+, but not p53-/- cells. Addition of Ix or Pb to either Ox, 5FU or Ir dramatically enhanced apoptosis in p53+/+ and -/- cells at doses that normally did not demonstrate any antitumor activity when administered as single agents. In p53 +/+ cells oxaliplatin alone induced 50% apoptosis at a concentration of 50 µM; Pp alone at 2 nM induced 25% apoptosis. However when IC50 of Ox was combined with 2nM Pp, we found a dramatic apoptosis-induction of >95%. This synergistic effect was less pronounced in p53-/- cells, however in p53-/- cells, addition of epothilones to standard chemotherapy also increased response rates efficiently compared to standard chemotherapy alone with 50µM Ox alone inducing 80% apoptosis in p53-/- cells. Similar intriguing results were seen for combinations of 5FU or Ir with Pb, as well as for combinations of standard chemotherapy with Ix. Thus, epothilones dramatically potentiate the efficacy of standard CRC chemotherapy (Ox, 5FU, Ir), and apoptosis induction is largely dependent on p53 status. These findings suggest that epothilones in combination with current standard chemotherapeutics in CRC may be clinically useful, and that evaluation of p53 status should be taken into consideration during the design of future clinical trials using these classes of agents. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation 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 2496.