The landscape of rare genetic variants in familial Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare hematological malignancy. Risk for WM is elevated 20-fold among first-degree relatives of patients with WM. However, the list of variants and genes that cause WM remains incomplete. In this study we analyzed exomes from 64 WM pedigrees for evidence of genetic susceptibility for this malignancy. We determined the frequency of pathogenic (P) or likely pathogenic (LP) variants among patients with WM; performed variant- and gene-level association analyses with the set of 166 WM cases and 681 unaffected controls; and examined the segregation pattern of deleterious variants among affected members in each pedigree. We identified P/LP variants in TREX1 and SAMHD1 (genes that function at the interface between innate immune response, genotoxic surveillance, and DNA repair) segregating in patients with WM from 2 pedigrees. There were additional P/LP variants in cancer-predisposing genes (eg, POT1, RECQL4, PTPN11, PMS2). In variant- and gene-level analyses, no associations were statistically significant after multiple testing correction. On a pathway level, we observed involvement of genes that play a role in telomere maintenance (q-value = 0.02), regulation of innate immune response (q-value = 0.05), and DNA repair (q-value = 0.08). Affected members of each pedigree shared multiple deleterious variants (median, n = 18), but the overlap between the families was modest. In summary, P/LP variants in highly penetrant genes constitute a modest proportion of the deleterious variants; each pedigree is largely unique in its genetic architecture, and multiple genes are likely involved in the etiology of WM.

Combined effects of exercise and immuno-chemotherapy treatments on tumor growth in MC38 colorectal cancer-bearing mice.

Acute exercise induces transient modifications in the tumor microenvironment and has been linked to reduced tumor growth along with increased infiltration of immune cells within the tumor in mouse models. In this study, we aimed to evaluate the impact of acute exercise before treatment administration on tumor growth in a mice model of MC38 colorectal cancer receiving an immune checkpoint inhibitor (ICI) and chemotherapy. Six-week-old mice injected with colorectal cancer cells (MC38) were randomized in 4 groups: control (CTRL), immuno-chemotherapy (TRT), exercise (EXE) and combined intervention (TRT/EXE). Both TRT and TRT-EXE received ICI: anti-PD1-1 (1 injection/week) and capecitabine + oxaliplatin (5 times a week) for 1 week (experimentation 1), 3 weeks (experimentation 2). TRT-EXE and EXE groups were submitted to 50 minutes of treadmill exercise before each treatment administration. Over the protocol duration, tumor size has been monitored daily. Tumor growth and microenvironment parameters were measured after the intervention on Day 7 (D7) and Day 16 (D16). From day 4 to day 7, tumor volumes decreased in the EXE/TRT group while remaining stable in the TRT group (p=0.0213). From day 7 until day 16 tumor volume decreased with no significant difference between TRT and TRT/EXE. At D7 the TRT/EXE group exhibited a higher total infiltrate T cell (p=0.0118) and CD8+ cytotoxic T cell (p=0.0031). At D16, tumor marker of apoptosis, vascular integrity and inflammation were not significantly different between TRT and TRT/EXE. Our main result was that acute exercise before immuno-chemotherapy administration significantly decreased early-phase tumor growth (D0 to D4). Additionally, exercise led to immune cell infiltration changes during the first week after exercise, while no significant molecular alterations in the tumor were observed 3 weeks after exercise.

A TLR4 Agonist Induces Osteosarcoma Regression by Inducing an Antitumor Immune Response and Reprogramming M2 Macrophages to M1 Macrophages.

Osteosarcoma (OsA) has limited treatment options and stagnant 5-year survival rates. Its immune microenvironment is characterized by a predominance of tumor-associated macrophages (TAMs), whose role in OsA progression remain unclear. Nevertheless, immunotherapies aiming to modulate macrophages activation and polarization could be of interest for OsA treatment. In this study, the antitumor effect of a liposome-encapsulated chemically detoxified lipopolysaccharide (Lipo-MP-LPS) was evaluated as a therapeutic approach for OsA. Lipo-MP-LPS is a toll-like receptor 4 (TLR4) agonist sufficiently safe and soluble to be IV administered at effective doses. Lipo-MP-LPS exhibited a significant antitumor response, with tumor regression in 50% of treated animals and delayed tumor progression in the remaining 50%. The agent inhibited tumor growth by 75%, surpassing the efficacy of other immunotherapies tested in OsA. Lipo-MP-LPS modulated OsA's immune microenvironment by favoring the transition of M2 macrophages to M1 phenotype, creating a proinflammatory milieu and facilitating T-cell recruitment and antitumor immune response. Overall, the study demonstrates the potent antitumor effect of Lipo-MP-LPS as monotherapy in an OsA immunocompetent model. Reprogramming macrophages and altering the immune microenvironment likely contribute to the observed tumor control. These findings support the concept of immunomodulatory approaches for the treatment of highly resistant tumors like OsA.

Contribution of Mesenchymal Stem Cells from Obese Adipose Tissue to PD-L1 Over-Expression and Breast Cancer Progression through Pathogenic Th17 Cell Activation.

BACKGROUND: Obesity is a well-known risk factor for cancer. We have previously reported the role of adipose-tissue-derived mesenchymal stem cells from obese individuals (ob-ASC) in the promotion of pathogenic Th17 cells and immune check point (ICP) upregulation. Thus, we postulated herein that this mechanism could contribute to breast cancer (BC) aggressiveness. METHODS: Conditioning medium (CM) from mitogen-activated ob-ASC and immune cell co-cultures were added to two human breast cancer cell line (BCCL) cultures. Expressions of pro-inflammatory cytokines, angiogenesis markers, metalloproteinases, and PD-L1 (a major ICP) were measured at the mRNA and/or protein levels. BCCL migration was explored in wound healing assays. Anti-cytokine neutralizing antibodies (Ab) were added to co-cultures. RESULTS: CM from ob-ASC/MNC co-cultures increased IL-1ß, IL-8, IL-6, VEGF-A, MMP-9, and PD-L1 expressions in both BCCLs and accelerated their migration. The use of Abs demonstrated differential effects for IL-17A and IFNγ on BCCL pro-inflammatory cytokine over-expression or PD-L1 upregulation, respectively, but potentiating effects on BCCL migration. Finally, co-cultures with ob-ASC, but not lean ASC, enhanced PD-L1 expression. CONCLUSIONS: Our results demonstrate increased inflammation and ICP markers and accelerated BCCL migration following the activation of pathogenic Th17 cells by ob-ASC, which could represent a new mechanism linking obesity with BC progression.

Antibody-drug conjugates come of age in oncology.

Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the potency of highly cytotoxic agents, potentially reducing the severity of side effects by preferentially targeting their payload to the tumour site. ADCs are being increasingly used in combination with other agents, including as first-line cancer therapies. As the technology to produce these complex therapeutics has matured, many more ADCs have been approved or are in late-phase clinical trials. The diversification of antigenic targets as well as bioactive payloads is rapidly broadening the scope of tumour indications for ADCs. Moreover, novel vector protein formats as well as warheads targeting the tumour microenvironment are expected to improve the intratumour distribution or activation of ADCs, and consequently their anticancer activity for difficult-to-treat tumour types. However, toxicity remains a key issue in the development of these agents, and better understanding and management of ADC-related toxicities will be essential for further optimization. This Review provides a broad overview of the recent advances and challenges in ADC development for cancer treatment.

A systemically administered detoxified TLR4 agonist displays potent antitumor activity and an acceptable tolerance profile in preclinical models.

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

Payload diversification: a key step in the development of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) is a fast moving class of targeted biotherapeutics that currently combines the selectivity of monoclonal antibodies with the potency of a payload consisting of cytotoxic agents. For many years microtubule targeting and DNA-intercalating agents were at the forefront of ADC development. The recent approval and clinical success of trastuzumab deruxtecan (Enhertu®) and sacituzumab govitecan (Trodelvy®), two topoisomerase 1 inhibitor-based ADCs, has shown the potential of conjugating unconventional payloads with differentiated mechanisms of action. Among future developments in the ADC field, payload diversification is expected to play a key role as illustrated by a growing number of preclinical and clinical stage unconventional payload-conjugated ADCs. This review presents a comprehensive overview of validated, forgotten and newly developed payloads with different mechanisms of action.

Clinical characteristics and outcome of 318 families with familial monoclonal gammopathy: A multicenter Intergroupe Francophone du Myélome study.

Familial forms of monoclonal gammopathy, defined as multiple myeloma (MM) or Monoclonal Gammopathy of Undetermined Significance (MGUS), are relatively infrequent and most series reported in the literature describe a limited number of families. MM rarely occurs in a familial context. MGUS is observed much more commonly, which can in some cases evolve toward full-blown MM. Although recurrent cytogenetic abnormalities have been described in tumor cells of sporadic cases of MM, the pathogenesis of familial MM remains largely unexplained. In order to identify genetic factors predisposing to familial monoclonal gammopathy, the Intergroupe Francophone du Myélome identified 318 families with at least two confirmed cases of monoclonal gammopathy. There were 169 families with parent/child pairs and 164 families with cases in at least two siblings, compatible with an autosomal transmission. These familial cases were compared with sporadic cases who were matched for age at diagnosis, sex and immunoglobulin isotype, with 10 sporadic cases for each familial case. The gender distribution, age and immunoglobulin subtypes of familial cases were unremarkable in comparison to sporadic cases. With a median follow-up of 7.4 years after diagnosis, the percentage of MGUS cases having evolved to MM was 3%. The median overall survival of the 148 familial MM cases was longer than that of matched sporadic cases, with projected values of 7.6 and 16.1 years in patients older and younger than 65 years, respectively. These data suggest that familial cases of monoclonal gammopathy are similar to sporadic cases in terms of clinical presentation and carry a better prognosis.

A pleiotropic variant in DNAJB4 is associated with multiple myeloma risk.

Pleiotropy, which consists of a single gene or allelic variant affecting multiple unrelated traits, is common across cancers, with evidence for genome-wide significant loci shared across cancer and noncancer traits. This feature is particularly relevant in multiple myeloma (MM) because several susceptibility loci that have been identified to date are pleiotropic. Therefore, the aim of this study was to identify novel pleiotropic variants involved in MM risk using 28 684 independent single nucleotide polymorphisms (SNPs) from GWAS Catalog that reached a significant association (P < 5 × 10-8 ) with their respective trait. The selected SNPs were analyzed in 2434 MM cases and 3446 controls from the International Lymphoma Epidemiology Consortium (InterLymph). The 10 SNPs showing the strongest associations with MM risk in InterLymph were selected for replication in an independent set of 1955 MM cases and 1549 controls from the International Multiple Myeloma rESEarch (IMMEnSE) consortium and 418 MM cases and 147 282 controls from the FinnGen project. The combined analysis of the three studies identified an association between DNAJB4-rs34517439-A and an increased risk of developing MM (OR = 1.22, 95%CI 1.13-1.32, P = 4.81 × 10-7 ). rs34517439-A is associated with a modified expression of the FUBP1 gene, which encodes a multifunctional DNA and RNA-binding protein that it was observed to influence the regulation of various genes involved in cell cycle regulation, among which various oncogenes and oncosuppressors. In conclusion, with a pleiotropic scan approach we identified DNAJB4-rs34517439 as a potentially novel MM risk locus.

The first ADC bearing the ferroptosis inducer RSL3 as a payload with conservation of the fragile electrophilic warhead.

The iron-dependent, non-apoptotic cell death, known as ferroptosis is an emerging strategy for the development of anticancer drugs. RSL3 was identified as an activator of ferroptosis through the inhibition of the glutathione peroxidase 4 (GPX4) which plays a crucial role in the cellular lipid oxidative stress. RSL3 is characterized by the presence of an electrophilic chloroacetyl moiety, namely warhead which covalently bonds to the catalytic and nucleophilic selenocysteine residue (Sec46) of GPX4. Like the major ferroptosis inducers, RSL3 suffers from lack of selectivity toward tumor cells. In this study, we report the first synthesis of an antibody-drug conjugate (ADC) containing RSL3 fragment and trastuzumab with the aim to deliver the agent selectively to tumors. The synthesis uses a judiciously chosen strategy to preserve the vital but fragile warhead. Full characterization of the ADC was accomplished, demonstrating the generation of a homogeneous DAR 8 conjugate. The robustness of the synthesis was successfully applied to another ADC associating the anti-CD74 mAb milatuzumab. The ADC induces ferroptotic cell death through reactive oxygen species accumulation and increases the activity of doxorubicin. The ADC associating trastuzumab and RSL3 may therefore offer potential applications in vectorized therapy alone or in combination with conventional chemotherapies.

In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti-PD-1/PD-L1 Therapies.

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti-PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti-PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti-PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

Analgesic and preventive effects of donepezil in animal models of chemotherapy-induced peripheral neuropathy: Involvement of spinal muscarinic acetylcholine M2 receptors.

BACKGROUND: Donepezil, a cholinesterase inhibitor approved in Alzheimer's disease, has demonstrated analgesic and preventive effects in animal models of oxaliplatin-induced neuropathy. To improve the clinical interest of donepezil for the management and prevention of chemotherapy-induced peripheral neuropathy (CIPN), a broader validation is required in different animal models of CIPN. METHODS: using rat models of CIPN (bortezomib, paclitaxel, and vincristine), the analgesic and preventive efficacies of donepezil were evaluated on tactile, cold and heat hypersensitivities. The involvement of muscarinic M2 acetylcholine receptors (m2AChRs) in analgesic effects was investigated at the spinal level. The absence of interference of donepezil with the cytotoxic effect of chemotherapy has been controlled in cancer cell lines. RESULTS: the analgesic efficacy of donepezil was demonstrated for all CIPN models, mainly on tactile hypersensitivity (maximal efficacy at 60 min, p < 0.05 vs. vehicle group). This effect was suppressed by an intrathecal injection of methoctramine (m2AChR antagonist). Regarding preventive effects, donepezil limited tactile hypersensitivity induced by paclitaxel, but not for other CIPN models. Donepezil did not modify the viability of cancer cells or the efficacy of anticancer drugs. CONCLUSIONS: donepezil had a broad analgesic effect on animal models of CIPN and this effect involved spinal m2AChRs. This work validates the repositioning of donepezil in the management of CIPN.

Proof of Concept: Protein Delivery into Human Erythrocytes Using Stable Cavitation.

Human erythrocytes represent candidates of choice as carriers for a wide range of drugs due to their unique biophysical and physiological properties. In this study, we used a sonoporation device generating and monitoring acoustic stable cavitation without any addition of contrast or nucleation agents. The device was evaluated for bovine serum albumin (BSA) delivery into human erythrocytes. After determining the adequate hematocrit percentage compatible with the generation of stable cavitation, we determined the optimal sonoporation conditions allowing BSA delivery while preserving erythrocyte integrity. Our results demonstrate that stable cavitation allows efficient delivery of proteins into human erythrocytes with limited lysis of these cells. In conclusion, our study allowed for the development of a stable and regulated cavitation program and the establishment of sonoporation conditions suitable for intracellular protein delivery while maintaining erythrocyte integrity. Additional investigations are needed to move from the proof of concept to a larger-scale application.

Variability in CD39 and CD73 protein levels in uveal melanoma patients.

Extracellular adenosine is produced from ATP by CD39 and CD73, and can modulate tumor development by acting on cancer cells or immune cells. Adenosine metabolism has been poorly studied in uveal melanoma. We studied the protein levels of CD39 and CD73 in a small, well described cohort of patients with uveal melanoma. Our results show a high variability in the levels of the two proteins, both in positivity and in intensity. Our results suggest that similar studies on larger cohorts could determine the clinical value and the druggability of these enzymes in the given clinical setting.Supplemental data for this article is available online at http://dx.doi.org/10.1080/15257770.2022.2032738.

A polygenic risk score for multiple myeloma risk prediction.

There is overwhelming epidemiologic evidence that the risk of multiple myeloma (MM) has a solid genetic background. Genome-wide association studies (GWAS) have identified 23 risk loci that contribute to the genetic susceptibility of MM, but have low individual penetrance. Combining the SNPs in a polygenic risk score (PRS) is a possible approach to improve their usefulness. Using 2361 MM cases and 1415 controls from the International Multiple Myeloma rESEarch (IMMEnSE) consortium, we computed a weighted and an unweighted PRS. We observed associations with MM risk with OR = 3.44, 95% CI 2.53-4.69, p = 3.55 × 10-15 for the highest vs. lowest quintile of the weighted score, and OR = 3.18, 95% CI 2.1 = 34-4.33, p = 1.62 × 10-13 for the highest vs. lowest quintile of the unweighted score. We found a convincing association of a PRS generated with 23 SNPs and risk of MM. Our work provides additional validation of previously discovered MM risk variants and of their combination into a PRS, which is a first step towards the use of genetics for risk stratification in the general population.

Impact of mouse model tumor implantation site on acquired resistance to anti-PD-1 immune checkpoint therapy.

Introduction: The use of tumor subcutaneous (SC) implantations rather than orthotopic sites is likely to induce a significant bias, in particular, in the field of immunotherapy. Methods: In this study, we developed and characterized MC38 models, implanted subcutaneously and orthotopically, which were either sensitive or rendered resistant to anti-PD1 therapy. We characterized the tumor immune infiltrate by flow cytometry at baseline and after treatment. Results and Discussion: Our results demonstrate several differences between SC and orthotopic models at basal state, which tend to become similar after therapy. These results emphasize the need to take into account tumor implantation sites when performing preclinical studies with immunotherapeutic agents.

Netrin-1 expression and targeting in multiple myeloma.

Deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5) receptors, play a key role in tumor progression of several solid tumors by inducing apoptosis when unbound to their ligand netrin-1. Netrin 1 is currently being evaluated as a therapeutic target. These receptors, known as dependence receptors, and their ligands, have not yet been extensively explored in hematological malignancies. Here, we performed a screening of various human myeloma cell lines and bone marrow samples from multiple myeloma patients for netrin-1 and its receptors to determine the expression of netrin 1 and its receptors in multiple myeloma as well as to assess the potential anti-myeloma activity of a novel anti-netrin-1 treatment (NP137). Our results showed heterogeneous expression of netrin-1 and its receptors DCC and UNC5H2(B) in six human myeloma lines. Additionally, immunohistochemistry and flow cytometry showed expression of these molecules in a majority of myeloma patient samples. In vitro NP137 did not induce apoptosis of myeloma cell lines yet enhanced the cytotoxicity of bortezomib and dexamethasone. In vivo, NP137 treatment of SCID mice with established RPMI8226 myeloma tumors led to a reduction of tumor size compared to controls. Ex vivo, NP137 lowered the plasma cells percentage in bone marrow aspirates in a fraction of the patient samples analyzed. These results suggest that netrin signaling could constitute a novel therapeutic target in multiple myeloma.