Petra Mayer

CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia

The cell-surface glycoprotein CD44 is expressed in chronic lymphocytic leukemia (CLL), but its functional role in this disease is poorly characterized. We therefore investigated the contribution of CD44 to CLL in a murine disease model, the Eµ-TCL1 transgenic mouse, and in CLL patients. Surface CD44 increased during murine CLL development. CD44 expression in human CLL was induced by stimulation with interleukin 4/soluble CD40 ligand and by stroma cell contact. Engagement of CD44 by its natural ligands, hyaluronic acid or chondroitin sulfate, protected CLL cells from apoptosis, while anti-CD44 small interfering RNAs impaired tumor cell viability. Deletion of CD44 during TCL1-driven murine leukemogenesis reduced the tumor burden in peripheral blood and spleen and led to a prolonged overall survival. The leukemic cells from these CD44 knockout animals revealed lower levels of antiapoptotic MCL1, a higher propensity to apoptosis, and a diminished B-cell receptor kinase response. The inhibitory anti-CD44 antibodies IM7 and A3D8 impaired the viability of CLL cells in suspension cultures, in stroma contact models, and in vivo via MCL1 reduction and by effector caspase activation. Taken together, CD44 expression in CLL is mediated by the tumor microenvironment. As a coreceptor, CD44 promotes leukemogenesis by regulating stimuli of MCL1 expression. Moreover, CD44 can be addressed therapeutically in CLL by specific antibodies.

Organometallic nucleosides induce non-classical leukemic cell death that is mitochondrial-ROS dependent and facilitated by TCL1-oncogene burden

BackgroundRedox stress is a hallmark of the rewired metabolic phenotype of cancer. The underlying dysregulation of reactive oxygen species (ROS) is interconnected with abnormal mitochondrial biogenesis and function. In chronic lymphocytic leukemia (CLL), elevated ROS are implicated in clonal outgrowth and drug resistance. The pro-survival oncogene T-cell leukemia 1 (TCL1) is causally linked to the high threshold towards classical apoptosis in CLL. We investigated how aberrant redox characteristics and bioenergetics of CLL are impacted by TCL1 and if this is therapeutically exploitable.MethodsBio-organometallic chemistry provided compounds containing a cytosine nucleobase, a metal core (ferrocene, ruthenocene, Fe(CO)3), and a 5’-CH2O-TDS substituent. Four of these metal-containing nucleoside analogues (MCNA) were tested for their efficacy and mode of action in CLL patient samples, gene-targeted cell lines, and murine TCL1-transgenic splenocytes.ResultsThe MCNA showed a marked and selective cytotoxicity towards CLL cells. MCNA activity was equally observed in high-risk disease groups, including those of del11q/del17p cytogenetics and of clinical fludarabine resistance. They overcame protective stromal cell interactions. MCNA-evoked PARP-mediated cell death was non-autophagic and non-necrotic as well as caspase- and P53-independent. This unconventional apoptosis involved early increases of ROS, which proved indispensible based on mitigation of MCNA-triggered death by various scavengers. MCNA exposure reduced mitochondrial respiration (oxygen consumption rate; OCR) and induced a rapid membrane depolarization (∆ΨM). These characteristics distinguished the MCNA from the alkylator bendamustine and from fludarabine. Higher cellular ROS and increased MCNA sensitivity were linked to TCL1 expression. The presence of TCL1 promoted a mitochondrial release of in part caspase-independent apoptotic factors (AIF, Smac, Cytochrome-c) in response to MCNA. Although basal mitochondrial respiration (OCR) and maximal respiratory capacity were not affected by TCL1 overexpression, it mediated a reduced aerobic glycolysis (lactate production) and a higher fraction of oxygen consumption coupled to ATP-synthesis.ConclusionsRedox-active substances such as organometallic nucleosides can confer specific cytotoxicity to ROS-stressed cancer cells. Their P53- and caspase-independent induction of non-classical apoptosis implicates that redox-based strategies can overcome resistance to conventional apoptotic triggers. The high TCL1-oncogenic burden of aggressive CLL cells instructs their particular dependence on mitochondrial energetic flux and renders them more susceptible towards agents interfering in mitochondrial homeostasis.

Actionable perturbations of damage responses by TCL1/ATM and epigenetic lesions form the basis of T-PLL

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution.T-cell prolymphocytic leukemia (T-PLL) is a rare malignancy with a poor prognosis. Here, the authors investigate the genomic landscape, gene expression profiles and functional mechanisms in 111 patients, highlighting TCL1 overexpression and ATM aberrations as core lesions which co-operate to impair DNA damage processing.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell–specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell–exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor– or CLL patient–derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971–84. ©2016 AACR.

AKT-pathway inhibition in chronic lymphocytic leukemia reveals response relationships defined by TCL1.

Cell survival in chronic lymphocytic leukemia (CLL) largely depends on B-cell receptor-induced AKT activation. Gain-of-function genomic lesions of PI3K-AKT-mTOR pathway components are usually absent in CLL. We previously established that a BCR-mediated growth response in CLL is determined by the oncogene T-cell leukemia 1 (TCL1) through a sensitizer effect on AKT phospho-activation. Despite high clinical response rates following AKT-cascade inhibition in CLL, resistances in a substantial proportion of patients call for reliable pre- and post-exposure strata to better predict compound responses. Using a panel of inhibitors with differential vertical affinities in the PI3K-AKT-mTOR axis, we describe distinct patterns and determinants of sensitivities in 75 CLL samples. The compounds specifically impacted the BCR-induced physical TCL1-AKT interaction. In general, there was an efficient and tumor-selective abrogation of cell survival in suspension or protective stromal-cell cultures. However, biochemical and survival responses were heterogeneous across CLL and showed only incomplete overlap across inhibitors. Sensitivity clusters could be defined by differential responses to selective pan-PI3K inhibition vs. compounds acting more down-stream. An elevated PI3K/AKT/mTOR activation state conferred sensitivity or resistance, depending on the applied inhibitor. In fact, down-stream interception by mTOR or dual mTOR/PI3K inhibition appears more efficient in cases expressing the BCR-response and poor-risk determinants of ZAP70 or TCL1. Finally, exploiting the TCL1-AKT interaction, peptide-based TCL1-interphase mimics were potent in steric AKT antagonization and in reducing CLL cell survival. Overall, this study provides informative response relationships in AKT-pathway interception that can help refining predictive models in BCR-pathway inhibition in CLL.

Abstract 370: T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling

The pathogenesis of the rare and aggressive T-cell prolymphocytic leukemia (T-PLL) is poorly understood, which particularly applies to a mechanistic concept around its hallmark oncogene TCL1A. Existing data implicate TCL1A as a catalytic enhancer of the oncogenic kinase AKT, a central node in a T-cell’s antigen receptor (TCR) signaling cascade, which mediates proliferation and differentiation. The levels and role of TCR activation in T-PLL’s pathogenesis are not known. To first clarify which physiological T-cell subset T-PLL cells most resemble, we performed comprehensive global gene expression profiling and immunophenotyping of primary T-PLL (n=79) in comparison to healthy-donor derived T-cell populations. Principle component analyses and gene signature alignments revealed a high similarity of T-PLL cells to (central) memory T-lymphocytes over naive T-cells. Surface markers revealed a spectrum of memory-type differentiation (n=69/79; 87%) with predominant central-memory stages (n=35/79; 44%). The usually TCR and/or CD28-coreceptor positive T-PLL cells revealed no restrictions to genetic or surface TCR-clonotypes. The abnormally high basal activation levels (surface CD25, CD38, CD69) correlated in their degree with inferior clinical outcomes (med. survival 20.8 vs 58.3 mo.; p=0.0012). In parallel, T-PLL cells lost expression of negative-regulatory TCR-co-receptors (e.g. CTLA-4, LAG3). Fittingly, TCR engagement of primary T-PLL cells revealed a trend to hyperactive intracellular responses and interleukin(IL)-2 release alongside a prominent Th1-cytokine program. T-PLL cells also showed a robust resistance to stimulation-induced cell death and agonistic CD95 ligation. TCR-derived signals (phospho-kinase induction, IL-2 release) were enhanced in vitro by the modulated presence of TCL1A with kinetics indicative of a sensitizer relationship, mainly in the CD3 axis as opposed to the CD28 branch. A mouse model with TCL1A-initiated protracted development of T-PLL (Lckpr-TCL1Atg) revealed congruent findings with the aberrant T-cell phenotype of human T-PLL. TCL1A expressing T-cells of this model, that were further equipped with monoclonal epitope-defined TCRs against ovalbumine or a chimeric-antigen-receptor (CAR) against carcinoembryonic antigen, gained a pre-leukemic growth advantage in scenarios of pulsed or continuous low-level receptor stimulation. Overall, we establish that T-PLL cells resemble antigen-experienced memory T-cells. Retention of functional effector responses to TCR stimulation and loss of restricting activation regulators underlie a highly activated phenotype and a marked resistance to death-inducing signals. TCL1A proactively enhances TCR responses and we postulate that this leukemogenic cooperation drives accumulation of memory-type cells that utilize amplified, hence permissive, low-level cognate antigen input. Note: This abstract was not presented at the meeting. Citation Format: Alexandra Schrader, Kathrin Warner, Sebastian Oberbeck, Giuliano Crispatzu, Petra Mayer, Sabine Putzer, Hans Diebner, Stephan Stilgenbauer, Georg Hopfinger, Jan Durig, Torsten Haferlach, Mark Lanasa, Ingo Roeder, Michael Hallek, Dimitrios Mougiakakos, Michael von Bergwelt-Baildon, Monika Bruggemann, Sebastian Newrzela, Hinrich Abken, Marco Herling. T-PLL cells resemble memory-type T-cells with aberrant effector functions implicating a leukemogenic cooperation of TCL1A and TCR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2017-370

New lessons learned in T-PLL: results from a prospective phase-II trial with fludarabine–mitoxantrone–cyclophosphamide–alemtuzumab induction followed by alemtuzumab maintenance

Abstract Clinical trials in T-cell prolymphocytic leukemia (T-PLL) are scarce. Based on a precursor study testing fludarabine, mitoxantrone, and cyclophosphamide followed by alemtuzumab (FMC-A), we aimed to improve this regimen by upfront combining subcutaneous (s.c.) alemtuzumab with FMC for four cycles followed by an alemtuzumab-maintenance (FMCA + A). This prospective multicenter phase-II trial assessed response, survival, and toxicity of that regimen administered to pretreated (n = 4) and treatment-naïve (n = 12) T-PLL patients. The best overall response rate after FMCA was 68.8% (n = 11) including five CRs (31.3%) and six PRs (37.5%). Six patients entered the alemtuzumab-maintenance. Median overall and progression-free survival was 16.7 and 11.2 months, respectively. Hematologic toxicities were the most frequent grade 3/4 side effects. A reduced incidence of CMV-reactivations was attributed to the prophylactic administration of valganciclovir. Overall, FMCA + A did not improve the efficacy of the FMC-A-regimen or of single i.v. alemtuzumab. It suggests that a chemotherapy backbone prevents efficient alemtuzumab dosing and confirms that intravenous alemtuzumab is to be preferred over its s.c. route in T-PLL. Trial registration: ClinicalTrials.gov identifier: NCT01186640.

Activity of the CD40 antagonistic antibody lucatumumab – insights from CLL-niche mimicking xenografts and fludarabine combinations

With great interest, we read the report by Byrd et al. [1] in this journal about lucatumumab (HCD122, formerly CHIR-12.12) in patients with relapsed chronic lymphocytic leukemia (CLL). This phase-I trial [1] concluded an acceptable tolerability and a limited single-agent activity of this CD40 monoclonal antibody (mAb). Two subsequent early-phase studies in relapsed/refractory (r/r) lymphoma [2] and multiple myeloma (MM) [3] confirmed selective response inductions as well as the encouraging safety profile and pharmacodynamics of this agent. We noted the interesting clinical response patterns of nodal debulking versus unaffected peripheral blood (PB) burden reported in CLL and specific lymphoma subsets, i.e., follicular lymphoma (FL). It prompted us to communicate here our novel preclinical observations in CLL and to review reported data on HCD122. Postulating a particular milieu-dictated activity and potential synergies in combined applications of HCD122, we addressed here the effects of lucatumumab at the regenerative niche of CLL and in drug combinations. In a first set of experiments, we employed our established NOD/SCID CLL-xenograft model.[4] The human disease engrafts in spleen without PB lymphocytosis and only when cotransplanted with T-/NK-cells and monocytes. This allows selective therapeutic and analytic access to CLL at its sites of active crosstalk with the CD40L-providing micromilieu while preserving prerequisites for antibody-dependent cellular cytotoxicity (ADCC). Freshly isolated PBMC from four patients with CLL were incubated ex vivo for 15 min with HCD122 (1 mg/kg and 10 mg/kg) or controls (PBS or hu-IgG1 isotype) followed by intravenous injection (100 10 cells) into 6-week-old female hosts (3 animals/patient). After 24 h, we observed a significant (paired t-tests) reduction of human leukocytes (CD45 flow cytometry) and CLL cells (CD5/19 gate) in the splenic suspensions at both HCD122 doses (CD45: HCD122 1 mg/kg: 0.29 ± 0.10, p 1⁄4 0.03; 10 mg/kg: 0.30 ± 0.19, p 1⁄4 0.03 (not shown); CLL cells: HCD122 1 mg/kg: 0.28 ± 0.07, p 1⁄4 0.02; 10 mg/kg: 0.28 ± 0.14, p 1⁄4 0.02 [Figure 1A]; all means ± SEMs). No reductions in splenic T-cell counts or (normal level) PB leukocytes (not shown) were observed. In a modification of this system, HCD122 treatment (days +14 and +21) was commenced after CLL cells had engrafted. Analysis of splenic infiltration at day +28 showed a significant depletion of human CLL cells in both HCD122 dosage groups (1 mg/kg: 0.72 ± 0.34, p 1⁄4 0.04; 10 mg/kg: 0.44 ± 0.19, p 1⁄4 0.03; means ± SEMs [Figure1B]). Although mainly attributable to leukemia cell reduction, the accompanying drop in infiltrating pan-leukocytes (CD45) was paralleled by decreases in infiltrating CD3 T-cells as well (not shown). A series of immunohistochemistries (IHC) on paraffin-embedded portions of these spleens confirmed the results semiquantitatively. The perivascular CLL infiltrates tended to be smaller in the HCD122-groups (as per H&E, CD45, CD19 [Figure 1C], and TCL1 stains), which was associated with slightly less-intense nuclear signals for NFkB-p65. There was no difference in CD38 (flow cytometry) as well as Ki67 and bcl2 signals (IHC). In another modification of the latter xenograft set-up in two additional patients (6 mice), the onset of splenic CLL cell depletion was as early as 2-h post-injection with peaks between 6–24 h. The next set of in vitro experiments on freshly isolated CLL patient cells showed that HCD122 reverses the CD40L-induced resistance to fludarabine cytotoxicity. An