Full chimaeric CAR.CIK from patients engrafted after allogeneic haematopoietic cell transplant: Feasibility, anti-leukaemic potential and alloreactivity across major human leukocyte antigen barriers.

Cytokine-induced killer lymphocytes (CIK) are a promising alternative to conventional donor lymphocyte infusion (DLI), following allogeneic haematopoietic cell transplantation (HCT), due to their intrinsic anti-tumour activity and reduced risk of graft-versus-host disease (GVHD). We explored the feasibility, anti-leukaemic activity and alloreactive risk of CIK generated from full-donor chimaeric (fc) patients and genetically redirected by a chimeric antigen receptor (CAR) (fcCAR.CIK) against the leukaemic target CD44v6. fcCAR.CIK were successfully ex-vivo expanded from leukaemic patients in complete remission after HCT confirming their intense preclinical anti-leukaemic activity without enhancing the alloreactivity across human leukocyte antigen (HLA) barriers. Our study provides translational bases to support clinical studies with fcCAR.CIK, a sort of biological bridge between the autologous and allogeneic sources, as alternative DLI following HCT.

Inhibition of bromodomain and extra-terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia.

The development of drugs able to target BTK, PI3k-delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non-recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19+ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax-resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first-line therapy in combination with venetoclax and as second-line therapy, after the emergence of venetoclax-resistant clones.

Identification of a new subclass of ALK-negative ALCL expressing aberrant levels of ERBB4 transcripts.

Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions.

The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA).

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR ß-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage.

High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

Expression of NKG2D and CD107 in CD8(+) effector memory lymphocytes in Churg-Strauss syndrome.

OBJECTIVES: Churg-Strauss syndrome (CSS) is a necrotising vasculitis of small vessels in which oligoclonally expanded TCR Vß CD8+ effector memory T cells populations (TEM) may be involved in vasculitic damage. The aim of this study was to assess the functional role of CD8+ T cells in CSS patients by flow cytometry analysis of membrane expression of cytotoxic markers NKG2D and CD107a. METHODS: Immunostaining of peripheral T cells and effector memory lymphocytes (TEM) from CSS patients and controls was performed by gating CD28 and CD45RA in the CD8+NKG2D+ and CD4+NKG2D+ populations. CD107a expression was evaluated in both whole CD8+ and CD4+ and the TEM cells by gating CD62 and CD45RA following polyclonal stimulation. RESULTS: NKG2D expression was shifted toward the CD8+CD28- fraction of T cells in CSS patients compared to healthy controls (56.1±25.8% versus 17.2±7.3%, respectively, p=0.002). CD8+Vß+ expanded T cells showed a significantly increased expression of NKG2D compared to the whole CD8+ T cell population (91.4±1.9% versus 79.7±3.8%, respectively, p=0.015). Moreover the CD8+ population from CSS upregulates CD107a on its surface upon polyclonal stimulation in a significantly higher proportion than healthy subjects (26.2±10.8% versus 8.2±2.9%, p=0.0031) and the majority CD8+ CD107+ cells from CSS patients showed a TEM phenotype compared to controls (64.8±4.9% vs. 19.8±2.9, respectively, p<0.001). CONCLUSIONS: In CSS, CD8+ TEM lymphocytes show markers of cytotoxic activity, which suggests a role for these cells in vasculitic damage.

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia.

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Aryloxyaryl Moiety.

In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.

Shedding Light on Targeting Chronic Myeloid Leukemia Stem Cells.

Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors (TKI). When TKI therapy is discontinued in CML patients in deep, sustained and apparently stable molecular remission, these cells in approximately half of the cases restart to grow, resuming the leukemic process. The elimination of these TKI resistant leukemic stem cells is therefore an essential step in increasing the percentage of those patients who can reach a successful long-term treatment free remission (TFR). The understanding of the biology of the LSCs and the identification of the differences, phenotypic and/or metabolic, that could eventually allow them to be distinguished from the normal hematopoietic stem cells (HSCs) are therefore important steps in designing strategies to target LSCs in a rather selective way, sparing the normal counterparts.

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Targeting Chronic Myeloid Leukemia Stem/Progenitor Cells Using Venetoclax-Loaded Immunoliposome.

CML is a hematopoietic stem-cell disorder emanating from breakpoint cluster region/Abelson murine leukemia 1 (BCR/ABL) translocation. Introduction of different TKIs revolutionized treatment outcome in CML patients, but CML LSCs seem insensitive to TKIs and are detectable in newly diagnosed and resistant CML patients and in patients who discontinued therapy. It has been reported that CML LSCs aberrantly express some CD markers such as CD26 that can be used for the diagnosis and for targeting. In this study, we confirmed the presence of CD26+ CML LSCs in newly diagnosed and resistant CML patients. To selectively target CML LSCs/progenitor cells that express CD26 and to spare normal HSCs/progenitor cells, we designed a venetoclax-loaded immunoliposome (IL-VX). Our results showed that by using this system we could selectively target CD26+ cells while sparing CD26- cells. The efficiency of venetoclax in targeting CML LSCs has been reported and our system demonstrated a higher potency in cell death induction in comparison to free venetoclax. Meanwhile, treatment of patient samples with IL-VX significantly reduced CD26+ cells in both stem cells and progenitor cells population. In conclusion, this approach showed that selective elimination of CD26+ CML LSCs/progenitor cells can be obtained in vitro, which might allow in vivo reduction of side effects and attainment of treatment-free, long-lasting remission in CML patients.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).

Nanocarriers as Magic Bullets in the Treatment of Leukemia.

Leukemia is a type of hematopoietic stem/progenitor cell malignancy characterized by the accumulation of immature cells in the blood and bone marrow. Treatment strategies mainly rely on the administration of chemotherapeutic agents, which, unfortunately, are known for their high toxicity and side effects. The concept of targeted therapy as magic bullet was introduced by Paul Erlich about 100 years ago, to inspire new therapies able to tackle the disadvantages of chemotherapeutic agents. Currently, nanoparticles are considered viable options in the treatment of different types of cancer, including leukemia. The main advantages associated with the use of these nanocarriers summarized as follows: i) they may be designed to target leukemic cells selectively; ii) they invariably enhance bioavailability and blood circulation half-life; iii) their mode of action is expected to reduce side effects. FDA approval of many nanocarriers for treatment of relapsed or refractory leukemia and the desired results extend their application in clinics. In the present review, different types of nanocarriers, their capability in targeting leukemic cells, and the latest preclinical and clinical data are discussed.

Bone marrow microenvironment: The guardian of leukemia stem cells.

Bone marrow microenvironment (BMM) is the main sanctuary of leukemic stem cells (LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease. Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.

Chronic myeloid leukemia stem cells.

Chronic myeloid leukemia (CML) is caused by BCRABL1 in a cell with the biological potential, intrinsic or acquired, to cause leukemia. This cell is commonly termed the CML leukemia stem cell (LSC). In humans a CML LSC is operationally-defined by ≥1 in vitro or in vivo assays of human leukemia cells transferred to immune-deficient mice. Results of these assays are sometimes discordant. There is also the unproved assumption that biological features of a CML LSC are stable. These considerations make accurate and precise identification of a CML LSC difficult or impossible. In this review, we consider biological features of CML LSCs defined by these assays. We also consider whether CML LSCs are susceptible to targeting by tyrosine kinase inhibitors (TKIs) and other drugs, and whether elimination of CML LSCs is needed to achieve therapy-free remission or cure CML.

An Ig Transmembrane Domain Motif Improves the Function of TCRs Transduced in Human T Cells: Implications for Immunotherapy.

Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and ß chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and ß chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.

Clonal CD8+ TCR-Vbeta expanded populations with effector memory phenotype in Churg Strauss syndrome.

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

Survivin-peptide vaccination elicits immune response after allogeneic nonmyeloablative transplantation: a safe strategy to enhance the graft versus tumor effect.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an adoptive immunotherapy strategy whose effectiveness relies on graft-versus-tumor (GVT) effect. We explored the feasibility of enhancing GVT after allo-HCT by peptide vaccination. Two myeloma patients were transplanted with a fludarabine-total body irradiation conditioning regimen and vaccinated with an HLA-A*0201-restricted modified survivin nonapeptide, plus montanide as adjuvant. At time of first vaccination, one patient had just attained serological remission despite documented relapse after transplant, while the other patient was in stable disease. Both patients had an immune response to vaccination: the frequency of survivin-specific CD8+ T cells increased between second and sixth vaccination and accounted for 0.5-0.8% of CD8+ cells; CD8+ cells were functional in ELISPOT assay. The first patient persists in complete remission with a follow-up of >5 years, while the second patient did not have a clinical response and vaccination was halted. We analyzed the T-cell receptor (TCR) repertoire of the first patient by spectratyping and found that vaccination did not affect the diversity of TCR profile, indicating that survivin clonotypes were probably spread in multiple TCR families. We generated a limited number (n = 4) of survivin-specific T cell clones: three were reactive only against the modified peptide, whereas one clone recognized also the naive peptide. Peptide vaccination is safe and applicable after allo-HCT and elicits an efficient antigen-specific T cell response without causing graft-versus-host disease.

Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5- a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors.

Human dihydroorotate dehydrogenase ( hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5- a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.

Tailoring CD19xCD3-DART exposure enhances T-cells to eradication of B-cell neoplasms.

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4+ cells into cytotoxic effectors required the presence of CD8+ cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.