Bone marrow Tfr2 deletion improves the therapeutic efficacy of the activin-receptor ligand trap RAP-536 in ß-thalassemic mice.

ß-thalassemia is a disorder characterized by anemia, ineffective erythropoiesis (IE), and iron overload, whose treatment still requires improvement. The activin receptor-ligand trap Luspatercept, a novel therapeutic option for ß-thalassemia, stimulates erythroid differentiation inhibiting the transforming growth factor ß pathway. However, its exact mechanism of action and the possible connection with erythropoietin (Epo), the erythropoiesis governing cytokine, remain to be clarified. Moreover, Luspatercept does not correct all the features of the disease, calling for the identification of strategies that enhance its efficacy. Transferrin receptor 2 (TFR2) regulates systemic iron homeostasis in the liver and modulates the response to Epo of erythroid cells, thus balancing red blood cells production with iron availability. Stimulating Epo signaling, hematopoietic Tfr2 deletion ameliorates anemia and IE in Hbbth3/+ thalassemic mice. To investigate whether hematopoietic Tfr2 inactivation improves the efficacy of Luspatercept, we treated Hbbth3/+ mice with or without hematopoietic Tfr2 (Tfr2BMKO/Hbbth3/+) with RAP-536, the murine analog of Luspatercept. As expected, both hematopoietic Tfr2 deletion and RAP-536 significantly ameliorate IE and anemia, and the combined approach has an additive effect. Since RAP-536 has comparable efficacy in both Hbbth3/+ and Tfr2BMKO/Hbbth3/+ animals, we propose that the drug promotes erythroid differentiation independently of TFR2 and EPO stimulation. Notably, the lack of Tfr2, but not RAP-536, can also attenuate iron-overload and related complications. Overall, our results shed further light on the mechanism of action of Luspatercept and suggest that strategies aimed at inhibiting hematopoietic TFR2 might improve the therapeutic efficacy of activin receptor-ligand traps.

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice.

Hepcidin, the main regulator of iron homeostasis, is repressed when erythropoiesis is acutely stimulated by erythropoietin (EPO) to favor iron supply to maturing erythroblasts. Erythroferrone (ERFE) has been identified as the erythroid regulator that inhibits hepcidin in stress erythropoiesis. A powerful hepcidin inhibitor is the serine protease matriptase-2, encoded by TMPRSS6, whose mutations cause iron refractory iron deficiency anemia. Because this condition has inappropriately elevated hepcidin in the presence of high EPO levels, a role is suggested for matriptase-2 in EPO-mediated hepcidin repression. To investigate the relationship between EPO/ERFE and matriptase-2, we show that EPO injection induces Erfe messenger RNA expression but does not suppress hepcidin in Tmprss6 knockout (KO) mice. Similarly, wild-type (WT) animals, in which the bone morphogenetic protein-mothers against decapentaplegic homolog (Bmp-Smad) pathway is upregulated by iron treatment, fail to suppress hepcidin in response to EPO. To further investigate whether the high level of Bmp-Smad signaling of Tmprss6 KO mice counteracts hepcidin suppression by EPO, we generated double KO Bmp6-Tmprss6 KO mice. Despite having Bmp-Smad signaling and hepcidin levels that are similar to WT mice under basal conditions, double KO mice do not suppress hepcidin in response to EPO. However, pharmacologic downstream inhibition of the Bmp-Smad pathway by dorsomorphin, which targets the BMP receptors, improves the hepcidin responsiveness to EPO in Tmprss6 KO mice. We concluded that the function of matriptase-2 is dominant over that of ERFE and is essential in facilitating hepcidin suppression by attenuating the BMP-SMAD signaling.

Erythroblast apoptosis and microenvironmental iron restriction trigger anemia in the VK*MYC model of multiple myeloma.

Multiple myeloma is a malignant disorder characterized by bone marrow proliferation of plasma cells and by overproduction of monoclonal immunoglobulin detectable in the sera (M-spike). Anemia is a common complication of multiple myeloma, but the underlying pathophysiological mechanisms have not been completely elucidated. We aimed to identify the different determinants of anemia using the Vk*MYC mouse, which spontaneously develops an indolent bone marrow localized disease with aging. Affected Vk*MYC mice develop a mild normochromic normocytic anemia. We excluded the possibility that anemia results from defective erythropoietin production, inflammation or increased hepcidin expression. Mature erythroid precursors are reduced in Vk*MYC bone marrow compared with wild-type. Malignant plasma cells express the apoptogenic receptor Fas ligand and, accordingly, active caspase 8 is detected in maturing erythroblasts. Systemic iron homeostasis is not compromised in Vk*MYC animals, but high expression of the iron importer CD71 by bone marrow plasma cells and iron accumulation in bone marrow macrophages suggest that iron competition takes place in the local multiple myeloma microenvironment, which might contribute to anemia. In conclusion, the mild anemia of the Vk*MYC model is mainly related to the local effect of the bone marrow malignant clone in the absence of an overt inflammatory status. We suggest that this reproduces the initial events triggering anemia in patients.

Disrupting pro-survival and inflammatory pathways with dimethyl fumarate sensitizes chronic lymphocytic leukemia to cell death.

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells.

IκBε deficiency accelerates disease development in chronic lymphocytic leukemia.

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options.

Longitudinal analysis of T cell receptor repertoires reveals shared patterns of antigen-specific response to SARS-CoV-2 infection.

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eµ-TCL1 mice through a CD40L-independent mechanism.

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eµ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP-/- mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L-/- mice, and TCL1+/+CD40-/- mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies.

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

Iron Induces Cell Death and Strengthens the Efficacy of Antiandrogen Therapy in Prostate Cancer Models.

PURPOSE: In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation. EXPERIMENTAL DESIGN: We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor-positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide. RESULTS: Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide-iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells.In vivo, iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide-iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion. CONCLUSIONS: Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy.