Chemotherapeutic agents have profound effects on cancer, stroma and immune cells that – in most cases – depend upon the dosage and schedule of administration. Preclinical and clinical studies ...summarized and discussed in the present review have demonstrated that maximum tolerable dosage (MTD) vs low-dosage, continuous (metronomic) administration of most chemotherapeutics have polarized effects on immune cells. In particular, metronomic schedules might be associated – among others effects – with activation of antigen presenting cells and generation of new T cell clones to enhance the activity of several types of immunotherapies. Ongoing and planned clinical trials in different types of cancer will confirm or dismiss this hypothesis and provide candidate biomarker data for the selection of patients who are likely to benefit from these combinatorial strategies.
•Metronomic chemotherapy has dose- and schedule-related, distinct effects over cancer, stroma and immune cells.•Combining metronomic chemotherapy with immunotherapy can enhance immune cell modulation for synergistic therapeutic benefits.•Intermittent metronomic chemotherapy activates APCs, generates stem cell-like T cell clones, to enhance the activity of ICIs.•Several clinical trials in different types of cancer are exploring these hypotheses.
Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical ...stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell-related effect. Single-cell transcriptome analysis of >50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1
stem-like CD8
T cells and altered the balance between terminally and progenitor-exhausted T cells favoring the latter. Overall, our data support the clinical investigation of this therapy in TNBC. SIGNIFICANCE: A combinatorial therapy in mouse models of breast cancer increases checkpoint inhibition by activating antigen-presenting cells, enhancing intratumoral Tcf1
stem-like CD8
T cells, and increasing progenitor exhausted CD8
T cells.
Abstract
Obesity increases risk for multiple tumors, but impact on leukemia and its underlying molecular mechanism are poorly understood. Studies from our group and others showed that risk and ...outcome of Acute Promyelocytic Leukemia (APL) are more strongly associated with obesity than other hematological neoplasms. Intriguingly, in 2 independent cohorts (Italian and TCGA), obesity was associated with ~4 fold higher incidence of internal tandem duplications of the leukemogenic kinase FLT3 (FLT3-ITD), the first example of obesity-associated genetic alterations in cancer (unpublished). Here we propose a molecular mechanisms underlying obesity-associated leukemogenesis through our studies in murine models. Exposure to High fat Diet (HFD), an established model of obesity-related pathology, strongly accelerated disease onset and mortality in 3 murine models of hematological cancer: PML-RARa knockin (PRKI), developing an APL-mimicking disease; FLT3ITD knockin (FIKI) developing myeloproliferation; a new double PRKI-FIKI knockin mouse, developing APL-like disease but, surprisingly, with delayed latency compared to PRKI, presumably due to the known exhaustion of hematopoietic stem cells (HSC) associated with FLT3ITD. Preleukemic HSCs from HFD PRKI mice showed increased DNA damage in Comet assays, but surprisingly this was not associated with increased mutational load as revealed by a novel whole genome sequencing-based method. Instead, HSCs derived from HFD PRKI mice showed enhanced self-renewal in in vitro replating assays. Similarly, in the FIKI model we observed enhanced engraftment in serial competitive transplantation experiments in HFD-fed recipients. Delving more deeply in the molecular mechanisms, through bioinformatic analysis of RNAseq data from the TCGA we found upregulation of the linoleic acid (LA) pathway in obese APL patients. LA is the richest fatty acid in HFD and in many western diets, thus a likely candidate for mediating HFD activity. LA significantly enhanced PRKI serial replating efficiency; this was abolished by pharmacologically blocking LA metabolism through a 5-lipoxygenase inhibitor or its transcriptional effects through a PPARd inhibitor. In the FIKI model and in a FLT3ITD-inducible cell line, we revealed a previously unappreciated induction of ER stress by FLT3ITD, likely to underlie FLT3ITD-induced HSC exhaustion. HFD (in vivo) and LA (in vitro) relieved FLT3ITD-induced ER stress, as revealed by the attenuation of markers of adaptive response to ER stress (Unfolded Protein Response and RNA IRE1-dependent RNA decay). In conclusion, our study suggests a novel and somewhat counterinuitive model for obesity-associated cancerogenesis: rather than inducing cellular stress, HFD-induced obesity enhances the self-renewal of preleukemic stem cells through LA-mediated alleviation of oncogene-induced proteotoxic stress
Citation Format: Luca Mazzarella, Paolo Falvo, Annagiulia Sanarico, Elena Gatti, Piergiuseppe Pelicci. Obesity favours leukemogenesis through enhanced preleukemic stem cell self renewal via polyunsaturated fatty acid-dependent ER stress relief abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 450.
MYC-over expressing lymphomas display inherent resistance to standard chemoimmunotherapy regimens and are characterised by genomic instability and constitutive activation of the DNA damage response ...(DDR) pathway. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate directed against CD19, a surface antigen broadly expressed in B-cell malignancies. Upon CD19 binding, Lonca induces DNA damage by delivering a pyrrolobenzodiazepine dimer-based, DNA crosslinking warhead into target cells. Here we hypothesised that DDR inhibition could enhance DNA damage induced by Lonca in CD19-positive lymphoma cells. Since MYC-overexpressing cells mainly rely on PARP activity to attenuate endogenous MYC-induced replicative stress, we combined Lonca with Talazoparib (an FDA-approved PARP inhibitor), in a panel of 12 DLBCL cell lines (7 with MYC-rearrangement), and in vivo in a MYC/BCL-2-rearranged DLBCL PDX model. Lonca and Talazoparib showed significant antiproliferative activity in a dose and time dependent manner in most cell lines at clinically achievable concentrations. The highest sensitivity to both compounds was observed in a BRCA2 mutant cell line (DOHH2). These data are in line with the known synthetic lethality between BRCA mutations and platinum derivatives and PARP inhibitors. The addition of Talazoparib significantly enhanced the antiproliferative effects of Lonca in DLBCL cell lines irrespective of MYC/BCL2 rearrangements and TP53 genomic alterations. Of note, the highest efficacy of the combination was observed in MYC-rearranged and MYC-overexpressing cell lines. The combination of Lonca and Talazo sinergistically increased DNA damage as evaluated by comet assay, induced enhanced cell death and caused specific changes in cell cycle dynamics. Enhanced apoptosis was confirmed by Annexin/PI staining and caspase 3/7 cleavage, assessed by caspase Glo assay and Western Blot analyses. As previously shown (Fusani et al., ASH 2021), while Lonca and Talazoparib affected cell cycle inducing G2/M phase arrest, the combination specifically increased the percentage of cells arrested in the BrdU negative S phase of cell cycle. Upon combined treatment, we observed increased DDR activation with the evaluation of several DDR biomarkers such as phospho(p)-ATM, pCHK2 Thr68, pCHK1 Ser345, gH2AX. To better understand the molecular mechanism underlying this synergistic interaction, we performed RNA-Seq analysis in two representative cell lines (OCI-LY18 and SUDHL5). Interestingly while Lonca did not cause significant changes in gene expression profiling (GEP) after 24 hours of incubation, the combination resulted in specific GEP changes such as down-regulation of histone H2B and aurora kinase A (AURKA), genes mainly involved in S and G2/M transition of the cell cycle. Similar results were obtained by combining Talazoparib and different PARP inhibitors with the alkylating agent cisplatin, indicating a class effect. The synergistic interaction of Lonca and Talazoparib was further confirmed in vivo in a MYC/BCL-2 double hit PDX model. As previously shown (Fusani et al., ASH 2021), Lonca/Talazoparib combination did not result in enhanced DNA damage induction in PBMC-derived B cells from healthy donors. Importantly, PBMC-derived T cells from healthy donors did not show any sign of DNA damage accumulation upon exposure to Lonca, Talazoparib and the combination. These data provide the rationale for future therapeutic strategies based on selective induction of DNA damage in neoplastic B cells in combination with DDR inhibition in aggressive MYC-positive B cell lymphoma.
Abstract
The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune ...system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs.
Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.
Group 2 innate lymphoid cells (ILC2s) are involved in human diseases, such as allergy, atopic dermatitis and nasal polyposis, but their function in human cancer remains unclear. Here we show that, in ...acute promyelocytic leukaemia (APL), ILC2s are increased and hyper-activated through the interaction of CRTH2 and NKp30 with elevated tumour-derived PGD2 and B7H6, respectively. ILC2s, in turn, activate monocytic myeloid-derived suppressor cells (M-MDSCs) via IL-13 secretion. Upon treating APL with all-trans retinoic acid and achieving complete remission, the levels of PGD2, NKp30, ILC2s, IL-13 and M-MDSCs are restored. Similarly, disruption of this tumour immunosuppressive axis by specifically blocking PGD2, IL-13 and NKp30 partially restores ILC2 and M-MDSC levels and results in increased survival. Thus, using APL as a model, we uncover a tolerogenic pathway that may represent a relevant immunosuppressive, therapeutic targetable, mechanism operating in various human tumour types, as supported by our observations in prostate cancer.Group 2 innate lymphoid cells (ILC2s) modulate inflammatory and allergic responses, but their function in cancer immunity is still unclear. Here the authors show that, in acute promyelocytic leukaemia, tumour-activated ILC2s secrete IL-13 to induce myeloid-derived suppressor cells and support tumour growth.
Abstract
Breast cancer represents a major clinical hurdle, with intra-tumor heterogeneity and metastatic outcome accounting for poor prognosis. Traditionally, its poly-clonal structure was ...investigated through the genetic barcoding of cells followed by the tracing of the offspring. However, this analysis does not allow a solid investigation of the transcriptional profile associated to clones throughout tumor progression. In this work, we exploited a recently published library of expressed barcodes to perform both clonal and transcriptional tracing in breast cancer by single-cell RNA sequencing. To this aim, human breast cancer cell line MDA-MB-231 was transduced with the above mentioned library and orthotopically transplanted in immune-deficient mice. To analyze our data we generated a pipeline aimed at identifying the barcodes at single-cell level and subsequently sequenced more than 20000 cells in six independent primary tumors. Our clonal distribution analysis strongly suggested that clones within the same tumor possess different growth potential. Furthermore, we found that breast tumors show a transcriptional heterogeneity characterized by two major transcriptional patterns: highly proliferating cells on one side, and stressed and dormant cells on the other. Notably, breast cancer clones were shown to belong to one of these two patterns, therefore suggesting that primary tumors are composed of both proliferative and non-proliferative clones. Interestingly, although the non-proliferative clones outnumbered the proliferative ones, the two different transcriptional patterns do not differ in terms of absolute number of cells. Analyses of breast cancer metastases to lungs and liver revealed that the clones that spread metastases belong to both the transcriptional patterns identified in the primary tumor. Interestingly, the metastatic clones were found to be rare in the primary tumors and shared between the two colonized organs. In addition, metastases recapitulated the same transcriptional heterogeneity of primary tumors, despite being composed of a much lower number of clones. Altogether, these findings represent an intriguing launching pad for the identification of the transcriptional markers whose inhibition may counter the metastatic outcome.
Citation Format: Niccolò Roda, Roman Hillje, Stefano Cheloni, Valentina Gambino, Giada Blandano, Paolo Falvo, Francesco Bertolini, Pier Giuseppe Pelicci. A transcriptional lineage tracing approach to unravel crucial gene expression patterns in breast cancer progression abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3127.
Abstract
Checkpoint inhibitors (CIs) instigate anticancer immunity, albeit only in a fraction of patients. Triple negative breast cancer (TNBC) is among the most aggressive and lethal BC types, and ...currently available therapies have an unsatisfactory clinical impact. The association of CIs with chemotherapy had encouraging results in randomized TNBC trials, but so far there has been no clear evidence of what should be considered the best combinatorial therapy. In B-cell non-Hodgkin's lympoma (NHL), CIs' clinical activity has been so far unsatisfactory. We designed the current study to define what could be considered the most efficient combinatorial regimen of chemotherapy plus CI in TNBC and NHL. We investigated different options including platinum (P), doxorubicin (D), taxol (T), vinorelbine (V), and cyclophosphamide ( C), i.e. the most effective chemotherapy drugs for these diseases. Capecitabine was not considered as found not to be effective in combination with CIs. As TNBC patients have a large intratumoral immune cell heterogeneity, we investigated two immune competent TNBC models: 4T1 with a predominant lymphoid intratumoral infiltrate and EMT6 with a predominant myeloid infiltrate. A C57BL/6 Eμ-myc transgenic NHL model, with a predominant lymphatic dissemination pattern, was also studied. We found that 1) Intermittent, medium-dosage C (C140), was more effective than other combinatorial regimens including the CI anti-PD1 plus low-dose C, or regular-dose T, D, or P. The association of V and anti-PD1 further increased the preclinical efficacy of C140; 2) both CD3+CD4+ and CD3+CD8+ T cells (at variance with other subsets of NK and myeloid cells) were needed to control local and metastatic neoplastic growth in mice treated with C140, V and anti-PD1. 3) The combinatorial therapy including C140, V and anti-PD1 was the most efficient in activating antigen presenting cells (APCs), and the investigation of TCR repertoire indicated that this therapy generated a significantly larger clonal expansion of both CD3+CD4+ and CD3+CD8+ T cells. 4) Single-cell transcriptome analysis of >50,000 intratumoural immune cells showed after C140, V and anti-PD1 therapy a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or an antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoural tcf1+ stem-like CD8+ T-cells and altered the balance between terminally and progenitor exhausted T-cells, favoring the latter. These data support the clinical investigation of this therapy in TNBC and NHL, and we are currently investigating the related transcriptional mechanisms inducing APC activation and T cell rewiring.
Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Giulia Mitola, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, PierGiuseppe Pelicci, Francesco Bertolini. A single-cell atlas of the effect of chemotherapeutics over intratumoral immune cells reveals that combining an alkylating agent and a vinca alkaloid can activate antigen presenting cells and increase tcf1+ stem-like CD8+ T-cells, thus improving anti-PD-1 efficacy in triple negative breast cancer and lymphoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1653.
Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting ...extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.
Abstract
Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, ...preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit.
Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.
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