A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

Purine Analogues Increase the Risk of Lethal and/or Prolonged COVID19 While Obinutuzumab Increases the Risk of Prolonged but Not Lethal Infection in Patients Treated for Lymphoid Malignancies -a Study of Krohem, the Croatian Group for Hematologic Diseases

Patients with lymphoid malignancies are at increased risk of death due to COVID19. Currently, it is not completely clear whether this is mainly due to disease biology or to anti-lymphoma treatment and whether the prognosis of infection differs in patients treated with different therapies. Anti-CD20 monoclonal antibodies increase the risk of prolonged infection. It is not known whether this risk is affected by the choice of the antibody. To study these questions, KroHem collected data on patients with lymphoid malignancies diagnosed with COVID19 between October 2020 and April 2021. Death during infection was considered as due to COVID19. Patients were considered to have prolonged disease if they were continuously or repetitively positive by PCR for more than 6 weeks. Percentage of patients with prolonged disease was calculated based on the number of patients with available data who were alive 6 weeks after beginning of infection. Treatment regimens were divided into those containing purine analogues (PA), mainly bendamustine and fludarabine, standard-dose chemotherapy without PA (e.g. CHOP, CVP, chlorambucil, etc.), high-dose chemotherapy without PA (e.g. DHAP, ICE, etc.), B-cell receptor inhibitors (iBCR) and venetoclax. We identified 314 patients, 20-88 years old (median 66), 180 male and 134 female; 75 were untreated, 61 off treatment and 178 on treatment (Table). Eleven (15%) untreated patients died; 10% had prolonged infection none of whom died. Ten (16%) off-treatment patients died; 9% had prolonged infection none of whom died. In the on-treatment group 6 (3%) are still prolonged positive, 110 (62%) recovered and 62 (35%) died; 42% had prolonged infection of whom 47% recovered and 42% died. The single allografted patient died as did both patients treated with CAR-T cells after prolonged infection. We analyzed prognostic factors for lethal and prolonged infection in the 175 conventionally treated patients. Disease type, use of anti-CD20 monoclonal antibodies, prior autologous stem-cell transplantation (ASCT) and line of treatment did not significantly affect mortality. Mortality was higher in older (p=0.0078) and those treated with PA in comparison to standard-dose chemotherapy without PA and iBCR (47% vs. 26%, p=0.012). The effect of anti-lymphoma therapy on mortality was similar in all age groups. All of the 7 patients who received neither cytotoxic agents nor iBCR (4 were on rituximab monotherapy, 2 on cyclosporine and 1 on vemurafenib) recovered, none had prolonged infection. Prolonged COVID19 was significantly more frequent in patients treated with anti-CD20 monoclonal antibodies (p=0.012), especially obinutuzumab (67% in comparison to 42% in those treated with rituximab and 21% in those treated without anti-CD20 antibodies). Treatment with PA also increased the risk of prolonged disease (69% vs. 25-45% in other groups, p=0.012). The effect of PA on prolonged infection was similar in patients treated with rituximab and obinutuzumab. Age, prior ASCT and treatment line did not significantly affect risk of prolonged infection. Our data suggest that the type of anti-lymphoma therapy is, besides age, a main determinant of prognosis of COVID19 in patients with lymphoid malignancies. Use of purine analogues, such as bendamustine and fludarabine, is related to increased risk of lethal and/or prolonged COVID19. These drugs should probably be avoided in patients with indolent NHL and CLL, diseases for whom other effective treatments are available, during the current pandemia. Anti-CD20 monoclonal antibodies seem to have a smaller effect on mortality, with obinutuzumab increasing the risk of prolonged disease, but not of death, in comparison to rituximab. Figure 1 Figure 1. Disclosures Aurer: takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Swixx/BMS: Honoraria; Teva/Pilva: Honoraria; Abbvie: Consultancy, Honoraria; sanofi genzyme: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Eusapharma: Consultancy, Honoraria. Jaksic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria. Mrdenovic: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Ostojic Kolonic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria. Lozic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria. Holik: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Novakovic Coha: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Bernes: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Krecak: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Moric Peric: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria. Mitrovic: Roche, Oktal-Pharma/Celtrion, Sandoz: Honoraria.

The Biology of Classic Hairy Cell Leukemia

Classic hairy cell leukemia (HCL) is a rare mature B-cell malignancy associated with pancytopenia and infectious complications due to progressive infiltration of the bone marrow and spleen. Despite tremendous therapeutic advances achieved with the implementation of purine analogues such as cladribine into clinical practice, the culprit biologic alterations driving this fascinating hematologic disease have long stayed concealed. Nearly 10 years ago, BRAF V600E was finally identified as a key activating mutation detectable in almost all HCL patients and throughout the entire course of the disease. However, additional oncogenic biologic features seem mandatory to enable HCL transformation, an open issue still under active investigation. This review summarizes the current understanding of key pathogenic mechanisms implicated in HCL and discusses major hurdles to overcome in the context of other BRAF-mutated malignancies.

A hajas sejtes leukémia korszerű diagnosztikája és kezelése

Összefoglaló. A hajas sejtes leukémia (HCL) egy indolens lefolyású ritka B-sejtes lymphoma. Diagnosztikájában jellegzetes morfológiai képén túlmenően a sejtek felszínén megtalálható markerek azonosítása áramlási citometriával, valamint a betegségben előforduló specifikus fehérjék immunhisztokémiai detektálása jelenti a rutineljárást. Kiemelt szerepet tölt be a differenciáldiagnosztikában a BRAF V600E mutációjának a kimutatása, melyre ma már számos módszer áll rendelkezésre, mint például az immunhisztokémia, pyroszekvenálás, allélspecifikus PCR vagy a droplet digitalis PCR. A tumorsejtek jelátviteli rendszerében és szabályozásában azonban a BRAF mutációjának következtében kialakuló folyamatos aktivitása mellett egyéb mechanizmusok is szerepet játszhatnak, többek között növekedési faktorok, interleukinek, adhéziós fehérjék vagy éppen mikro-RNS-ek. A patomechanizmus egyre részletesebb megismerése érdekében egyéb daganatokhoz hasonlóan a HCL-ben is aktív kutatások folynak a genetikai háttér feltérképezésére új generációs szekvenálás segítségével. Leírtak már nagy százalékban előforduló mutációkat a CDKN1B-, KLF2- és KMT2C-gének esetében, továbbá egyéb génekben is alacsonyabb előfordulási aránnyal. Genetikailag, sőt klinikai manifesztáció és terápiás válasz alapján is jelentős eltérések láthatóak a klasszikus és variáns HCL-es betegek között, elkülönítésük épp ezért rendkívül fontos. Míg a klasszikus esetben első vonalban alkalmazott purin nukleozid analógok kiemelkedő válaszreakciót képesek kiváltani, a variáns HCL-es betegek gyakran refrakterek a kezelésre, és esetükben a célzott BRAF-gátlók szintén hatástalanok. Számos klinikai kutatás zajlik a jelenleg is alkalmazott terápiás szerek optimalizálása, kombinációban történő alkalmazása, valamint egyéb lymphoid daganatokban alkalmazott gyógyszerek és új támadáspontok ellen tervezett molekuláknak a HCL kezelésébe történő bevonása céljából. Summary. Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy. In addition to characteristic morphology of HCL cells, the identification of the cellular surface markers and the expression of specific proteins by flow cytometry and immunohistochemistry are routine procedure in HCL diagnosis. Detection of BRAF V600E mutation plays key role in differential diagnosis of HCL which can be detected by several novel methods, such as immunohistochemistry, pyrosequencing, allele specific PCR or droplet digital PCR. Beside the BRAF mutation there can be other mechanisms causing constitutive activity in the signaling pathway and regulating the tumor cells such as growth factors, interleukins, adhesion proteins and micro-RNAs as well. Like in other malignancies, in order to clarify the pathomechanism, the genetic background of HCL is also actively investigated by next-generation sequencing. Frequent mutations were described in CDKN1B, KLF2 and KMT2C genes, moreover in other genes with lower incidence rate, as well. Genetically, and even in clinical manifestation and therapeutic response, significant differences can be found between patients with classical and variant HCL. While classical type has outstanding response for the first-line treatment with purine analogues, patients with variant HCL are often refractory to the treatment, and the BRAF inhibitors prove to be ineffective. Therefore, it is really important to distinguish these two entities. Several clinical studies are still in progress for the optimization and application of combining the currently applied therapeutic agents, furthermore other drugs that used in lymphoid malignancies are under investigation. New target molecules are also designed as novel therapeutic opportunity in HCL treatment.

Key genetic elements, single and in clusters, underlying geographically dependent SARS-CoV-2 genetic adaptation and their impact on binding affinity for drugs and immune control

Objectives To define key genetic elements, single or in clusters, underlying SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) evolutionary diversification across continents, and their impact on drug-binding affinity and viral antigenicity. Methods A total of 12 150 SARS-CoV-2 sequences (publicly available) from 69 countries were analysed. Mutational clusters were assessed by hierarchical clustering. Structure-based virtual screening (SBVS) was used to select the best inhibitors of 3-chymotrypsin-like protease (3CL-Pr) and RNA-dependent RNA polymerase (RdRp) among the FDA-approved drugs and to evaluate the impact of mutations on binding affinity of these drugs. The impact of mutations on epitope recognition was predicted following Grifoni et al. (Cell Host Microbe 2020; 27 671–80.) Results Thirty-five key mutations were identified (prevalence: ≥0.5%), residing in different viral proteins. Sixteen out of 35 formed tight clusters involving multiple SARS-CoV-2 proteins, highlighting intergenic co-evolution. Some clusters (including D614GSpike + P323LRdRp + R203KN + G204RN) occurred in all continents, while others showed a geographically restricted circulation (T1198KPL-Pr + P13LN + A97VRdRp in Asia, L84SORF-8 + S197LN in Europe, Y541CHel + H504CHel + L84SORF-8 in America and Oceania). SBVS identified 20 best RdRp inhibitors and 21 best 3CL-Pr inhibitors belonging to different drug classes. Notably, mutations in RdRp or 3CL-Pr modulate, positively or negatively, the binding affinity of these drugs. Among them, P323LRdRp (prevalence: 61.9%) reduced the binding affinity of specific compounds including remdesivir while it increased the binding affinity of the purine analogues penciclovir and tenofovir, suggesting potential hypersusceptibility. Finally, specific mutations (including Y541CHel + H504CHel) strongly hampered recognition of Class I/II epitopes, while D614GSpike profoundly altered the structural stability of a recently identified B cell epitope target of neutralizing antibodies (amino acids 592–620). Conclusions Key genetic elements reflect geographically dependent SARS-CoV-2 genetic adaptation, and may play a potential role in modulating drug susceptibility and hampering viral antigenicity. Thus, a close monitoring of SARS-CoV-2 mutational patterns is crucial to ensure the effectiveness of treatments and vaccines worldwide.

The incidence, risk factors and outcomes of chemotherapy related acute kidney injury in China.

e24161 Background: Nephrotoxicity of chemotherapeutic agents remains a significant complication limiting the efficacy of the treatment. However, comprehensive data on the epidemiology and outcomes of chemotherapy related acute kidney Injury in China is lacking. Methods: We conducted a nationwide cohort study of hospitalized patients from 25 general and children’s hospitals in China during 2013-2015. Patient-level data were obtained from the electronic hospitalization information system, prescription database and laboratory databases of all cancer patients who received chemotherapy and had at least two serum creatinine tests within any 7-day window during the hospitalization. AKI was defined and staged according to Kidney Disease Improving Global Outcomes criteria. The incidence of AKI in patients with various type of cancer and chemotherapeutic agents was examined. The outcomes of AKI, including in-hospital mortality, death after discharge, kidney recovery, and length of stay, were also assessed. Results: A total of 23,232 cancer patients, including 3,120 children ( < 18 years old), 16,310 adult (19-65 years old) and 3,802 elderly patients ( > 65 years old), were analyzed. Platinum compounds and pyrimidine analogues were the most common used chemotherapy agents for cancer patients. The overall incidence of AKI was 4.9%. Patients with urinary system malignancy (12.3%), hematological malignancy (10.2%) and nerve motor system malignancy (4.1%) have the highest incidence of HA-AKI. The top three types of chemotherapy drugs with the highest incidence of AKI were Purine analogues (30.1%), folic acid analogues (15.3%) and combinations of antineoplastic agents (14.1%). The nephrotoxicity of chemotherapy drugs was different among age groups. AKI is associated with a higher risk of in-hospital mortality and death after discharge. Conclusions: The risk of AKI in cancer patients varied in different age group, type of cancer and chemotherapeutic agents.