Effects In Leukemia Cell Lines Research Articles

Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the β-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

First-in-Human CD4 CAR Clinical Trial on Peripheral T-Cell Lymphoma

Background CD19 CAR T cell therapy has achieved success in treating acute lymphoblastic leukemia. However, the treatment for Sezary syndrome, an aggressive form of cutaneous peripheral T cell lymphoma (PTCL) has remained a challenge. Despite patients with Sezary syndrome typically receiving multiple treatments within their disease progression, the prognosis is poor with 5 year survival rate of only 24%. Therefore, it is crucial to establish a novel treatment for PTCLs. CD4 is uniformly expressed on most mature T cell lymphoma, which makes it a promising target for treating PTCLs. Here, we present the efficacy of CD4 CAR T cell in our preclinical study and the success in level 1 dose escalation clinical trial on patients with Sezary syndrome. Methods We engineered a CD4 CAR with scFv (single-chain variable fragment) with CD28 and 4-1BB co-activators fused to CD3zeta and a leader sequence of CD8. The efficacy of CD4 CAR was tested with CD4+ leukemic cell line, primary CD4+ PTCL patient samples and multiple mouse models. An alemtuzumab safety switch has also been established to ensure the elimination of CAR T cells following tumor eradication. Children and adults with PTCLs were enrolled in our phase 1 dose escalation trial to evaluate the safety and efficacy of CD4 CAR T cell antitumor activity. Results Coculture assays results showed that CD4 CAR T cells displayed profound tumor killing effects in leukemia cell lines, primary patient samples and multiple mouse model systems. Our preclinical findings suggest that CD4 CAR T cells is an effective approach in treating PTCLs. Patients enrolled in the phase 1 dose escalation trial have shown remarkable response to CD4 CAR T cells treatment. Noticeably, a 54-yr-old patient diagnosed with Sezary syndrome had achieved complete remission with CD4 CAR T cell therapy. Prior to admission, he had been having symptoms of erythroderma, pruritus and scaling of the skin for over 10 years and had been resistant to multiple lines of chemotherapy. Before the initiation of CAR therapy, patient's body skin has extensive leukemia infiltrate (Fig. 1A) confirmed with skin biopsy (Fig. 1B) with bone marrow and blood comprising 50% leukemic cells (Fig. 1C). Patient received a total dose of 3x10^6 /kg single dose CAR T cells, following which fluconazole and valacyclovir were administrated for infection prophylaxis. Since patient received CD4 CAR T cell infusion, the percentage of CAR T cells (Fig 1. D) in peripheral blood had continue to increase as well as NK cells. (Fig 1. E) On day 13, patient had achieved complete remission with the percentage of leukemia cells in blood decreased to zero (Fig. 1C). On day 28, the appearance of the skin had undergone drastic change from what was before the treatment. Noticeable skin regeneration on both legs of the patients was observed (Fig 1. F). Flow cytometry of bone marrow and peripheral blood confirmed the absence of tumor cells. In addition, Skin biopsy on multiple sites demonstrated absence of leukemia infiltrates post CAR treatment (Fig. 1G). Patient was subsequently discharged with no additional medication needed. Throughout the treatment, patient had developed no infections with Grade II CRS toxicity noted. No other toxicities were observed. Updated results on other patients enrolled in this clinical trial including adverse events will be presented. Conclusion Our first-in-human clinical trial demonstrates promising efficacy of CD4 CAR T cell therapy in treating patients with refractory Sezary syndrome. cCAR is able to eradicate leukemia blasts, exerting a profound tumor killing effect that is superior to traditional chemotherapies. Disclosures Pinz: iCell Gene Therapeutics LLC: Employment. Ma:iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Consultancy, Equity Ownership, Research Funding; iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

Abstract 214: Anticancer effect of Compound A, a novel modulator of the glucocorticoid receptor, in acute leukemia cells

Abstract Glucocorticoids (GCs) are widely used in chemotherapy of blood cancers, including childhood acute leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, many adverse metabolic effects of glucocorticoids are mediated by GR transactivation that requires GR binding to hormone-responsive elements in gene promoters. Thus, selective GR activators (SEGRA) that modify GR, shift GR functions towards transrepression resulting in improved therapeutic index, hold a great potential for the GR-targeted chemotherapies. Recently we and others characterized novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride or Compound A (CpdA), and showed that CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer properties: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion. Here we report that CpdA at concentrations 10−6-10−5M exerted cytostatic (evaluated by MTT test) and cytotoxic (evaluated by FACS analysis of DNA fragmentation as sub-G1 peak in propidium iodide-labeled cells) effects in K564, NB-4 and CEM leukemia cell lines. We found that these cell lines express wild type nonmutated GR. Consequently, these cell lines present a good model system for the further comparative analysis of CpdA and GCs effects. Leukemia cell lines were sensitive to apoptotic effect of both GR ligands: Dexamethasone (Dex) and CpdA. Dex induced apoptosis at the concentrations 10−7 to 10−5M; and CpdA affected cell viability at concentrations 5×10−6−10−5M. As a first step towards the evaluation of toxicological profile of this promising anti-cancer compound, we assessed its mutagenicity using Ames test. CpdA was not mutagenic either in frameshift or in base-substitution indicator strains, TA98 and TA100, respectively, at the wide range of concentrations (10−9-10−4M). Overall, our data suggest that novel non-mutagenic GR modulator has a high chemotherapeutic potential. The detailed mechanisms of its anti-cancer effects in leukemia cell lines is under investigation. 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 214.

Abstract 3684: Developing STAT3 protein inhibitors as adjuvant therapeutics: Promising synergistic effects in human cancers

Abstract Signal Transducer and Activator of Transcription 3 (Stat3) protein is a cytosolic transcription factor that relays signals from receptors in the plasma membrane directly to the nucleus, and is routinely hyper-activated in many human cancers and diseases. STAT3 induces anti-apoptotic gene expression programs (e.g. Bcl-xL) and the over-expression of cell cycle regulators (e.g. cyclin D1) that contribute significantly to the resistance of cancer to current chemotherapeutic strategies. Since most cancer drugs aim to initiate apoptosis, tumor cells containing activated STAT3 have an intrinsic resistance to current treatment strategies. It has therefore been postulated that STAT3 inhibitors could play a significant role in the future of adjuvant cancer therapies by sensitizing human tumors to traditional chemotherapy. By examining the protein-protein interaction interface and employing computational modeling, we have thus developed a series of small molecule inhibitors of the transcriptionally active STAT3-STAT3 homo-dimer complex. Lead inhibitors showed potent anti-STAT3 activity in vitro and in tumor cell lines, as well as in malignant cells taken from leukemia patients. More specifically, these compounds displayed single digit micromolar activity against breast, prostate, pancreatic and leukemia cell lines and showed negligible cytotoxic effects on healthy cells treated with high µM concentrations of compounds. Preliminary adjuvant studies with a series of clinically relevant therapeutics have shown impressive synergistic effects in leukemia cell lines, as well as in patient tumor cells. 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 3684.