ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

Abstract Background Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. Methods The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. Results Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. Conclusions Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases. Graphical Abstrac

Frequency and Targeted Detection of HLA-DPB1 T Cell Epitope Disparities Relevant in Unrelated Hematopoietic Stem Cell Transplantation

The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by die presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB I donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions. (c) 2007 American Society for Blood and Marrow Transplantation The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by die presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB I donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions. (c) 2007 American Society for Blood and Marrow Transplantation Inflammation and immune reaction, or pre-existing immunity towards commonly used viral vectors for gene therapy severely impair long-term gene expression in the central nervous system (CNS), impeding the possibility to repeat the therapeutic intervention. Here, we show that injection of a helper-dependent adenoviral (HD-Ad) vector by lumbar puncture into the cerebrospinal fluid (CSF) of non-human primates allows long-term (three months) infection of neuroepithelial cells, also in monkeys bearing a pre-existing anti-adenoviral immunity. Intrathecal injection of the HD-Ad vector was not associated with any sign of systemic or local toxicity, nor by signs of a CNS-specific immune reaction towards the HD-Ad vector. Injection of HD-Ad vectors into the CSF circulation may thus represent a valuable approach for CNS gene therapy allowing for long-term expression and re-administration

Therapeutic and diagnostic applications of minor histocompatibility antigen HA-1 and HA-2 disparities in allogeneic hematopoietic stem cell transplantation: a survey of different populations.

Minor histocompatibility antigens (mHags) HA-1 and HA-2 are encoded by biallelic loci, with immunogenic variants, HA-1(H) and HA-2(V), which induce strong HLA-A2-restricted alloreactive T-cell responses, and nonimmunogenic counterparts, HA-1(R) and HA-2(M), which represent functional null alleles that are poorly presented by HLA class I molecules. HA-1 and HA-2 are potential targets of selective graft-versus-leukemia,e and graft-versus-tumor reactivity after allogeneic hematopoietic stem cell transplantation (HSCT); however, these applications are restricted to a limited number of patients. Here, we show that a far more frequent application of HA-1 and HA-2 disparity relies on their use as markers for the state of host chimerism after allogeneic HSCT. We have determined allelic frequencies of 29.3% and 70.7% for HA-1(H) and HA-1(R), respectively, and of 83.7% and 16.3% for HA-2(V) and HA-2(M), respectively, in > 200 healthy individuals from northern Italy. Similar frequencies were observed in nearly 100 patients affected by hematologic malignancies or solid tumors, thus showing that HA-1 and HA-2 variabilitv are not associated with the presence of cancer. On the basis of these data, we predict that HA-1 and HA-2 can be used in 32.8% and 23.5% of Italian transplant patients, respectively, as markers for the state of host chimerism, whereas exploitation of disparity for these mHags for targeted immunotherapy will be possible in 10.7% and 1.1% of Italian patients, respectively. Retrospective HA-2 typing of bone marrow., aspirates obtained from a patient during complete remission or recurrence of acute myeloid leukemia after haploidentical HSCT showed the feasibility of using HA-2 as a surrogate marker for disease monitoring. Because of an apparent north-south gradient for HA-1 allelic frequencies, with higher frequencies for the HA-1(H) variant reported in white populations from Southern Europe as compared with Northern Europe and North America, the diagnostic applicability of HA-1 disparity will be slightly more frequent in transplant patients from the north. Taken together, our data show that determination of HA-1 and HA-2 variability can be an important parameter for the selection of allogeneic stem cell donors, in particular for patients affected by hematologic malignancies without a tumor-specific molecular marker. (c) 2006 American Society for Blood and Marrow Transplantation