Yihui Ma

PP2A-activating drugs selectively eradicate tki-resistant chronic myeloid leukemic stem cells

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase-independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression--but not activity--of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase-independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1-positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Antitumor effects of OSU-2S, a nonimmunosuppressive analogue of FTY720, in hepatocellular carcinoma†‡

Accumulating evidence suggests the therapeutic potential of the immunosuppressive agent FTY720 (fingolimod) in hepatocellular carcinoma (HCC). Based on our previous finding that FTY720 mediates apoptosis in HCC cells by activating reactive oxygen species (ROS)–protein kinase Cδ (PKCδ) signaling independent of effects on sphingosine‐1‐phosphate (S1P) receptors, we embarked on the pharmacological exploitation of FTY720 to develop a nonimmunosuppressive analogue with antitumor activity. This effort led to the development of OSU‐2S, which exhibits higher potency than FTY720 in suppressing HCC cell growth through PKCδ activation. In contrast to FTY720, OSU‐2S was not phosphorylated by sphingosine kinase 2 (SphK2) in vitro, and did not cause S1P1 receptor internalization in HCC cells or T lymphocyte homing in immunocompetent mice. Although devoid of S1P1 receptor activity, OSU‐2S exhibited higher in vitro antiproliferative efficacy relative to FTY720 against HCC cells without cytotoxicity in normal hepatocytes. Several lines of pharmacological and molecular genetic evidence indicate that ROS–PKCδ–caspase‐3 signaling underlies OSU‐2S–mediated antitumor effects, and that differences in the antitumor activity between FTY720 and OSU‐2S were attributable to SphK2‐mediated phosphorylation of FTY720, which represents a metabolic inactivation of its antitumor activity. Finally, OSU‐2S exhibited high in vivo potency in suppressing xenograft tumor growth in both ectopic and orthotopic models without overt toxicity. Conclusion: Using the molecular platform of FTY720, we developed OSU‐2S, a novel PKCδ‐targeted antitumor agent, which is devoid of S1P1 receptor activity and is highly effective in suppressing HCC tumor growth in vivo. These findings suggest that OSU‐2S has clinical value in therapeutic strategies for HCC and warrants continued investigation in this regard. (HEPATOLOGY 2011;)

A Potent Indole-3-Carbinol–Derived Antitumor Agent with Pleiotropic Effects on Multiple Signaling Pathways in Prostate Cancer Cells

Indole-3-carbinol has emerged as a promising chemopreventive agent due to its in vivo efficacy in various animal models. However, indole-3-carbinol exhibits weak antiproliferative potency and is unstable in acidic milieu. Thus, this study was aimed at exploiting indole-3-carbinol to develop potent antitumor agents with improved chemical stability. This effort culminated in OSU-A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol}, which is resistant to acid-catalyzed condensation, and exhibits 100-fold higher apoptosis-inducing activity than the parent compound. Relative to indole-3-carbinol, OSU-A9 displays a striking qualitative similarity in its effects on the phosphorylation or expression of multiple signaling targets, including Akt, mitogen-activated protein kinases, Bcl-2 family members, survivin, nuclear factor-kappaB, cyclin D1, p21, and p27. The ability of OSU-A9 to concurrently modulate this broad range of signaling targets underscores its in vitro and in vivo efficacy in prostate cancer cells. Nevertheless, despite this complex mode of mechanism, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9 than PC-3 and LNCaP prostate cancer cells. Treatment of athymic nude mice bearing established s.c. PC-3 xenograft tumors with OSU-A9 at 10 and 25 mg/kg i.p. for 42 days resulted in a 65% and 85%, respectively, suppression of tumor growth. Western blot analysis of representative biomarkers in tumor lysates revealed significant reductions in the intratumoral levels of phosphorylated (p-) Akt, Bcl-xL, and RelA, accompanied by robust increases in p-p38 levels. In conclusion, the ability of OSU-A9 to target multiple aspects of cancer cell survival with high potency suggests its clinical value in prostate cancer therapy.

Development of p21 Activated Kinase-Targeted Multikinase Inhibitors That Inhibit Thyroid Cancer Cell Migration

CONTEXT The p21 activated kinases (PAKs) are a family of serine/threonine kinases that are downstream effectors of small GTPase Cdc42 and Rac. PAKs regulate cell motility, proliferation, and cytoskeletal rearrangement. PAK isoform expression and activity have been shown to be enhanced in cancer and to function as an oncogene in vivo. PAKs also have been implicated in cancer progression. OBJECTIVE In thyroid cancer, we have previously determined that PAK overactivation is common in the invasive fronts of aggressive tumors and that it is functionally involved in thyroid cancer cell motility using molecular inhibitors. We report the development of two new PAK-inhibiting compounds that were modified from the structure OSU-03012, a previously identified multikinase inhibitor that competitively blocks ATP binding of both phosphoinositide-dependent kinase 1 (PDK1) and PAK1. RESULTS Seventeen compounds were created by combinatorial chemistry predicted to inhibit PAK activity with reduced anti-PDK1 effect. Two lead compounds were identified based on the ability to inhibit PAK1 activity in an ATP-competitive manner without discernible in vivo PDK1 inhibitory activity in thyroid cancer cell lines. Both compounds reduced thyroid cancer cell viability. Although they are not PAK-specific on a multikinase screening assay, the antimigration activity effect of the compounds in thyroid cancer cells was rescued by overexpression of a constitutively active PAK1, suggesting this activity is involved in this biological effect. CONCLUSIONS We have developed 2 new multikinase inhibitors with anti-PAK activity that may serve as scaffolds for further compound development targeting this progression-related thyroid cancer target.

OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway

Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU‐DY7, a novel D‐tyrosinol derivative targeting p38 mitogen‐activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC‐1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose‐ and time‐dependent manner. The OSU‐DY7‐induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase‐3 activation and poly (ADP‐ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z‐VAD‐FMK. Interestingly, OSU‐DY7‐induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B‐CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU‐DY7‐mediated cytotoxicity. Furthermore, OSU‐DY7‐induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU‐DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.

Abstract LB-109: BCR-ABL1 kinase activity but not its expression is dispensable for Ph+ quiescent stem cell survival which depends on the PP2A-controlled Jak2 activation and is sensitive to FTY720 treatment

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background : The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods : HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assays; Western blot, confocal microscopy and FACS analyses. Results : We observed increased BCR-ABL1 expression with impaired kinase activity in quiescent CML HSCs, in which BCR-ABL1 per se is required for induction of JAK2 that subsequently activated β-catenin and inhibited PP2A. In fact, PP2A was suppressed in CML but not normal CD34+/CD38−/CD90+ HSCs. FTY720 and/or its non-immunosuppressive ( S )-FTY720-OMe derivative markedly reduced survival and self-renewal of CML but not normal quiescent HSCs through BCR-ABL1 kinase-independent and PP2A-mediated JAK2 and β-catenin inhibition. Importantly, FTY720 also strongly diminished BCR-ABL1+ LT-HSC frequency in serial BM transplantation assays. Conclusions : The pharmacologic targeting of the newly-identified BCR-ABL1 kinase-independent JAK2/β-catenin interplay in quiescent HSCs with FTY720 and its derivatives, might lead to cessation of lifelong patient dependence on TKIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-109. doi:10.1158/1538-7445.AM2011-LB-109