Allogeneic, donor-derived, second-generation, CD19-directed CAR-T cells for the treatment of pediatric relapsed/refractory BCP-ALL.

Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T cells transduced with a second-generation (4.1BB) CD19-directed CAR for treatment of patients with BCP-ALL in a hospital-exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR-Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy-based manufacturing process (CD19-CAR-Lenti_ALLO). Thirteen children/young adults received ALLO-CAR-T cells between March 2021 and October 2022. Doses ranged between 1.0 × 106 and 3.0 × 106 CAR-T cells per kg. The toxicity profile was comparable with that of autologous CAR-T cells, characterized mainly by cytopenia, cytokine release syndrome (maximum grade 1), and grade 2 immune-effector cell-associated neurotoxicity syndrome. One case of acute graft-versus-host disease (GVHD) occurred and was rapidly controlled with steroids and ruxolitinib. None of the other patients, including 3 given ALLO-CAR-T cells from an HLA-haploidentical donor, experienced GVHD. Two patients received ALLO-CAR-T cells before HSCT and showed a significant expansion of CAR-T cells without any sign of GVHD. All patients obtained complete remission (CR) with absence of minimal residual disease in the bone marrow. With a median follow-up of 12 months (range, 5-21), 8 of 13 patients maintained CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly refractory BCP-ALL relapsing after allo-HSCT without showing increased toxicity as compared with autologous CAR-T cells.

GD2-CART01 for Relapsed or Refractory High-Risk Neuroblastoma.

BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. METHODS: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). RESULTS: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. CONCLUSIONS: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).

PI3K/Akt Pathway: The Indestructible Role of a Vintage Target as a Support to the Most Recent Immunotherapeutic Approaches.

Pathologic activation of PI3Ks and the subsequent deregulation of its downstream signaling pathway is among the most frequent events associated with cellular transformation, cancer, and metastasis. PI3Ks are also emerging as critical factors in regulating anti-tumor immunity by either promoting an immunosuppressive tumor microenvironment or by controlling the activity and the tumor infiltration of cells involved in the immune response. For these reasons, significant pharmaceutical efforts are dedicated to inhibiting the PI3K pathway, with the main goal to target the tumor and, at the same time, to enhance the anti-tumor immunity. Recent immunotherapeutic approaches involving the use of adoptive cell transfer of autologous genetically modified T cells or immune check-point inhibitors showed high efficacy. However, mechanisms of resistance to these kinds of therapy are emerging, due in part to the inhibition of effector T cell functions exerted by the immunosuppressive tumor microenvironment. Here, we first describe how inhibition of PI3K/Akt pathway contribute to enhance anti-tumor immunity and further discuss how inhibitors of the pathway are used in combination with different immunomodulatory and immunotherapeutic agents to improve anti-tumor efficacy.

Recent advances in searching c-Myc transcriptional cofactors during tumorigenesis.

BACKGROUND: The mechanism by which c-Myc exerts its oncogenic functions is not completely clear and different hypotheses are still under investigation. The knowledge of the capacity of c-Myc to bind exclusively E-box sequences determined the discrepancy between, on the one hand, genomic studies showing the binding of c-Myc to all active promoters and, on the other hand, the evidence that only 60% or less of the binding sites have E-box sequences. MAIN BODY: In this review, we provide support to the hypothesis that the cooperation of c-Myc with transcriptional cofactors mediates c-Myc-induced cellular functions. We produce evidence that recently identified cofactors are involved in c-Myc control of survival mechanisms of cancer cells. CONCLUSION: The identification of new c-Myc cofactors could favor the development of therapeutic strategies able to compensate the difficulty of targeting c-Myc.

Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens.

Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens.

Co-targeting of Bcl-2 and mTOR pathway triggers synergistic apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia.

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL.

Two types of BCR interactions are positively selected during leukemia development in the Eµ-TCL1 transgenic mouse model of CLL.

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Eµ-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL.

Purification and characterization of adipose-derived stem cells from patients with lipoaspirate transplant.

Techniques for medical tissue regeneration require an abundant source of human adult stem cells. There is increasing evidence that adipose stem cells contribute to restoration of tissue vascularization and organ function. The object of our study was to isolate and characterize adult adipose-derived stem cells from patients undergoing on lipoaspirate transplant with the aim to improve tissue regeneration. Adipose-derived stem cells were isolated and purified from the lipoaspirate of 15 patients and characterized for CD markers and the ability to differentiate toward the adipogenic lineage. We found that purified adipose stem cells express high level of CD49d, CD44, CD90, CD105, CD13, and CD71 and these markers of staminality were maintained at high level for at least 3 months and seven passages of in vitro culture. As expected, these cells resulted negative for the endothelial and hematopoietic-specific markers CD31, CD106, CD34, and CD45. Differentiation towards adipogenic lineage demonstrated that purified adipose-derived stem cells are still able to become adipocytes at least 3 months after in vitro culture. The analysis of Akt and MAPK phosphorylation confirmed a modulation of their activity during differentiation. Interestingly, we established for the first time that, among the p53 family members, a strong upregulation of p63 expression occurs in adipocytic differentiation, indicating a role for this transcription factor in adipocytic differentiation. Taken together, these data indicate that purified lipoaspirate-derived stem cells maintain their characteristic of staminality for a long period of in vitro culture, suggesting that they could be applied for cell-based therapy to improve autologous lipoaspirate transplant.

Lymphoid EVA1 expression is required for DN1-DN3 thymocytes transition.

BACKGROUND: Thymus organogenesis and T lymphocyte development are accomplished together during fetal life. Proper development and maintenance of thymus architecture depend on signals generated by a sustained crosstalk between developing thymocytes and stromal elements. Any maturation impairment occurring in either cellular component leads to an aberrant thymic development. Gene expression occurring during T lymphocyte differentiation must be coordinated in a spatio-temporal fashion; one way in which this is achieved is through the regulation by cell-cell adhesion and interactions. PRINCIPAL FINDINGS: We examined the role played by Epithelial V-like Antigen 1 (EVA1), an Ig adhesion molecule expressed on thymus epithelial cells (TEC) and immature thymocytes, in T cell development by employing RNA interference in vitro and in vivo models. Fetal liver derived haematopoietic progenitors depleted of Eva1, displayed a delayed DN1-DN3 transition and failed to generate CD4CD8 double positive T cells in OP9-DL1 coculture system. In addition, we could observe a coordinated Eva1 up-regulation in stromal and haematopoietic cells in coculture control experiments, suggesting a possible EVA1 involvement in TEC-haematopoietic cells crosstalk mechanisms. Similarly, Rag2-gamma c double knock out mice, transplanted with Eva1 depleted haematopoietic progenitors displayed a 10-fold reduction in thymus reconstitution and a time delayed thymocytes maturation compared to controls. CONCLUSIONS: Our findings show that modulation of Eva1 expression in thymocytes is crucial for lymphocyte physiological developmental progression and stromal differentiation.

MDM2-regulated degradation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis.

In response to DNA damage, p53 induces either cell-cycle arrest or apoptosis by differential transcription of several target genes and through transcription-independent apoptotic functions. p53 phosphorylation at Ser46 by HIPK2 is one determinant of the outcome because it takes place only upon severe, nonrepairable DNA damage that irreversibly drives cells to apoptosis. Here, we show that p53 represses its proapoptotic activator HIPK2 via MDM2-mediated degradation, whereas a degradation-resistant HIPK2 mutant has increased apoptotic activity. Upon cytostatic, nonsevere DNA damage, inhibition of HIPK2 degradation is sufficient to induce p53Ser46 phosphorylation and apoptosis, converting growth-arresting stimuli to apoptotic ones. These findings establish HIPK2 as an MDM2 target and support a model in which, upon nonsevere DNA damage, p53 represses its own phosphorylation at Ser46 due to HIPK2 degradation, supporting the notion that the cell-cycle-arresting functions of p53 include active inhibition of the apoptotic ones.

Repression of the antiapoptotic molecule galectin-3 by homeodomain-interacting protein kinase 2-activated p53 is required for p53-induced apoptosis.

Galectin 3 (Gal-3), a member of the beta-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

ATM is activated by default in mitosis, localizes at centrosomes and monitors mitotic spindle integrity.

We previously showed that ATM is responsible for p53 phosphorylation at Ser15 and localization at centrosomes during mitosis. When p53 centrosomal localization is prevented by inhibiting polymerization of spindle microtubules, a stabilized form of p53 is transmitted to daughter cells that arrest in the next G(1) phase of the cell cycle after exit from mitosis. AT cells are unable to both localize p53 at centrosomes in mitosis and arrest after exposure to mitotic-spindle poisons. Here we show that during mitosis ATM is activated by phosphorylation at Ser1981 and localizes at centrosomes. When mitotic spindle is disrupted by nocodazole, ATM is displaced from centrosomes and colocalizes with phospho-Ser15-p53 under the form of spots dispersed in the mitotic cytoplasm. After release from nocodazole-block, as soon as cells exit mitosis, p53 is redirected to the nucleus and its Ser15 phosphorylation is substituted by phosphorylation at Ser46. We suggest that ATM is activated by default at each mitotic onset and phosphorylates p53 at Ser15 so as to keep it inactive at centrosomes when the spindle is correctly in place or, in case of inactivation of the mitotic spindle, to maintain the memory of a perturbed mitosis.

Identification of an aberrantly spliced form of HDMX in human tumors: a new mechanism for HDM2 stabilization.

The HDMX protein is closely related to HDM2 with which it shares different structural domains, particularly the p53 binding domain and the ring finger domain, where the two HDM proteins interact. Several oncogenic forms derived from splicing of HDM2 have been described in cancer. This work aimed at investigating whether analogous forms of HDMX exist in human tumors. Here, we report the characterization of an aberrantly spliced form of HDMX, HDMX211, isolated from the thyroid tumor cell line, ARO. HDMX211 binds and stabilizes the HDM2 protein. Although it lacks the p53 binding domain, HDMX211 also stabilizes p53 by counteracting its degradation by HDM2. However, the resulting p53 is transcriptionally inactive and increasingly associated to its inhibitor HDM2. Expression of HDMX211 strongly enhances the colony-forming ability of human cells in the presence or absence of wild-type p53. Conversely, depletion of HDMX211 by small interfering RNA significantly reduces the growth of ARO cells and increases their sensitivity to chemotherapy. Screening of lung cancer biopsies shows the presence of HDMX211 in samples that overexpress HDM2 protein, supporting a pathologic role for this new protein. This is the first evidence of a variant form of HDMX that has oncogenic potential independently of p53. HDMX211 reveals a new mechanism for overexpression of the oncoprotein HDM2. Most interestingly, it outlines a possible molecular explanation for a yet unclarified tumor phenotype, characterized by simultaneous overexpression of HDM2 and wild-type p53.

Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin.

An emerging paradigm holds that loss of negative signalling to receptor tyrosine kinases (RTKs) is permissive for their oncogenic activity. Herein, we have addressed tumor suppression by RALT/MIG-6, a transcriptionally controlled feedback inhibitor of ErbB RTKs, in breast cancer cells. Knockdown of RALT expression by RNAi enhanced the EGF-dependent proliferation of normal breast epithelial cells, indicating that loss of RALT signalling in breast epithelium may represent an advantageous condition during ErbB-driven tumorigenesis. Although mutational inactivation of the RALT gene was not detected in human breast carcinomas, RALT mRNA and protein expression was strongly and selectively reduced in ERBB2-amplified breast cancer cell lines. Reconstitution of RALT expression in ERBB2-amplified SKBr-3 and BT474 cells inhibited ErbB-2-dependent mitogenic signalling and counteracted the ability of ErbB ligands to promote resistance to the ErbB-2-targeting drug Herceptin. Thus, loss of RALT expression cooperates with ERBB2 gene amplification to drive full oncogenic signalling by the ErbB-2 receptor. Moreover, loss of RALT signalling may adversely affect tumor responses to ErbB-2-targeting agents.