Optimizing drug combinations for T-PLL: restoring DNA damage and P53-mediated apoptotic responses.

ABSTRACT: T-prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm associated with marked chemotherapy resistance and continued poor clinical outcomes. Current treatments, that is, the CD52-antibody alemtuzumab, offer transient responses, with relapses being almost inevitable without consolidating allogeneic transplantation. Recent more detailed concepts of T-PLL's pathobiology fostered the identification of actionable vulnerabilities: (1) altered epigenetics, (2) defective DNA damage responses, (3) aberrant cell-cycle regulation, and (4) deregulated prosurvival pathways, including T-cell receptor and JAK/STAT signaling. To further develop related preclinical therapeutic concepts, we studied inhibitors of histone deacetylases ([H]DACs), B-cell lymphoma 2 (BCL2), cyclin-dependent kinase (CDK), mouse double minute 2 (MDM2), and classical cytostatics, using (1) single-agent and combinatorial compound testing in 20 well-characterized and molecularly profiled primary T-PLL (validated by additional 42 cases) and (2) 2 independent murine models (syngeneic transplants and patient-derived xenografts). Overall, the most efficient/selective single agents and combinations (in vitro and in mice) included cladribine, romidepsin ([H]DAC), venetoclax (BCL2), and/or idasanutlin (MDM2). Cladribine sensitivity correlated with expression of its target RRM2. T-PLL cells revealed low overall apoptotic priming with heterogeneous dependencies on BCL2 proteins. In additional 38 T-cell leukemia/lymphoma lines, TP53 mutations were associated with resistance toward MDM2 inhibitors. P53 of T-PLL cells, predominantly in wild-type configuration, was amenable to MDM2 inhibition, which increased its MDM2-unbound fraction. This facilitated P53 activation and downstream signals (including enhanced accessibility of target-gene chromatin regions), in particular synergy with insults by cladribine. Our data emphasize the therapeutic potential of pharmacologic strategies to reinstate P53-mediated apoptotic responses. The identified efficacies and their synergies provide an informative background on compound and patient selection for trial designs in T-PLL.

Diagnostic management of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in close interaction with therapeutic considerations.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare malignancy derived from plasmacytoid dendritic cells, can mimic both acute leukemia and aggressive T-cell lymphoma. Therapy of this highly aggressive hematological disease should be initiated as soon as possible, especially in light of novel targeted therapies that have become available. However, differential diagnosis of BPDCN remains challenging. This retrospective study aimed to highlight the challenges to timely diagnoses of BPDCN. We documented the diagnostic and clinical features of 43 BPDCN patients diagnosed at five academic hospitals from 2001-2022. The frequency of BPDCN diagnosis compared to AML was 1:197 cases. The median interval from the first documented clinical manifestation to diagnosis of BPDCN was 3 months. Skin (65%) followed by bone marrow (51%) and blood (45%) involvement represented the most common sites. Immunophenotyping revealed CD4 + , CD45 + , CD56 + , CD123 + , HLA-DR + , and TCL-1 + as the most common surface markers. Overall, 86% (e.g. CD33) and 83% (e.g., CD7) showed co-expression of myeloid and T-cell markers, respectively. In the median, we detected five genomic alterations per case including mutational subtypes typically involved in AML: DNA methylation (70%), signal transduction (46%), splicing factors (38%), chromatin modification (32%), transcription factors (32%), and RAS pathway (30%), respectively. The contribution of patients (30%) proceeding to any form of upfront stem cell transplantation (SCT; autologous or allogeneic) was almost equal resulting in beneficial overall survival rates in those undergoing allogeneic SCT (p = 0.0001). BPDCN is a rare and challenging entity sharing various typical characteristics of other hematological diseases. Comprehensive diagnostics should be initiated timely to ensure appropriate treatment strategies.

Accurate extrinsic and intrinsic peak broadening modelling for time-resolved in situ ball milling reactions via synchrotron powder X-ray diffraction.

The debate on the mechanisms which underpin mechanochemical reactions via ball mill grinding is still open. Our ability to accurately measure the microstructural (crystal size and microstrain) evolution of materials under milling conditions as well as their phase composition as a function of time is key to the in-depth understanding of the kinetics and driving forces of mechanochemical transformations. Furthermore, all ball milling reactions end with a steady state or milling equilibrium - represented by a specific phase composition and relative microstructure - that does not change as long as the milling conditions are maintained. The use of a standard sample is essential to determine the instrumental contribution to the X-ray powder diffraction (XRPD) peak broadening for time-resolved in situ (TRIS) monitoring of mechanochemical reactions under in operando conditions. Using TRIS-XRPD on a ball milling setup, coupled with low-energy synchrotron radiation, we investigated different data acquisition and analysis strategies on a silicon standard powder. The diffraction geometry and the microstructural evolution of the standard itself have been studied to model the instrumental contribution to XRPD peak broadening throughout the grinding activity. Previously proposed functions are here challenged and further developed. Importantly, we show that minor drifts of the jar position do not affect the instrumental resolution function significantly. We here report and discuss the results of such investigations and their application to TRIS-XRPD datasets of inorganic and organic ball mill grinding reactions.

Outcomes of second allogeneic stem cell transplantation and anti-relapse strategies in patients with relapsed/refractory acute myeloid leukemia: A unicentric retrospective analysis.

Second allogeneic stem cell transplantation (allo-SCT2) represents a rescue option for selected patients (pts) with relapsed/refractory (r/r) acute myeloid leukemia (AML). Still, relapse rates post-allo-SCT2 remain high and effective anti-relapse strategies and predictive biomarkers remain to be defined. We here analyzed a cohort of 41 AML patients (pts) undergoing allo-SCT2 in our center. Allo-SCT2 induced a third hematologic complete remission (CR) in 37 pts, at costs of a 36% non-relapse mortality rate. Furthermore, 19 pts eventually relapsed post allo-SCT2. Addressing relapse after allo-SCT2, 14 pts (74%) underwent cell-based anti-relapse strategies, including third allogeneic transplantation (allo-SCT3; 3/14), donor lymphocyte infusions (DLIs) combined with either 5-azacytidin and venetoclax (4/14) or chemotherapeutic agents (7/14). Notably, six of seven pts (86%) who received either allo-SCT3 or a combination therapy of DLIs, 5-azacytidine and venetoclax achieved CR despite poor cytogenetics post-allo-SCT2 (e.g., TP53). Finally, 11 of 41 pts were alive at the last follow-up (seven CR2, three CR3, one partial remission) resulting in estimated 2- and 5-year overall survival of 35% and 25%, respectively.

Bis-isonicotinoyl linkers containing polyaromatic scaffolds: synthesis, structure and spectroscopic properties.

In this study, a new series of extended linkers containing different polyaromatic chromophores (biphenyl, naphthalene, anthracene, fluorene, 9,9-dimethylfluorene and fluorenone) functionalized with isonicotinoyl moieties have been synthesized by Pd-catalyzed cross-coupling reactions involving isonicotinamide and the appropriate aromatic dibromide. The optimized protocol led to the isolation of the target molecules in good yield and with high purity. These were characterized by 1H NMR, FTIR, MS, and elemental analysis and their solid state structures were solved by single-crystal X-ray diffraction analysis. Electronic absorption and emission spectra were collected both in solution (DMF) and in the solid state. TDDFT calculations were carried out to investigate the effect of the isonicotinoyl moieties on the spectral features of the central chromophores. Although in solution only the linker containing a fluorenone scaffold shows a weak fluorescence, all the isolated linkers turned out to be fluorescent in the solid state, thus paving the way for their use for the fabrication of fluorescent MOFs.

Functional apoptosis profiling identifies MCL-1 and BCL-xL as prognostic markers and therapeutic targets in advanced thymomas and thymic carcinomas.

BACKGROUND: Multi-omics studies have shown a high and lack of common driver mutations in most thymomas (TH) and thymic carcinomas (TC) that hamper the development of novel treatment approaches. However, deregulation of apoptosis has been proposed as a common hallmark of TH and TC. BH3 profiling can be utilized to study the readiness of living cancer cells to undergo apoptosis and their dependency on pro-survival BCL-2 family proteins. METHODS: We screened a cohort of 62 TH and TC patient samples for expression of BCL-2 family proteins and used the TC cell line 1889c and native TH for dynamic BH3 profiling and treatment with BH3 mimetics. RESULTS: Immunohistochemical overexpression of MCL-1 and BCL-xL was a strong prognostic marker of TH and TC, and BH3 profiling indicated a strong dependency on MCL-1 and BCL-xL in TH. Single inhibition of MCL-1 resulted in increased binding of BIM to BCL-xL as an escape mechanism that the combined inhibition of both factors could overcome. Indeed, the inhibition of MCL-1 and BCL-xL in combination induced apoptosis in a caspase-dependent manner in untreated and MCL-1-resistant 1889c cells. CONCLUSION: TH and TC are exquisitely dependent on the pro-survival factors MCL-1 and BCL-xL, making them ideal candidates for co-inhibition by BH3 mimetics. Since TH show a heterogeneous dependency on BCL-2 family proteins, upfront BH3 profiling could select patients and tailor the optimal therapy with the least possible toxicity.

Synthesis, characterization, antimicrobial and cytotoxic activity and DNA-binding properties of d-metal complexes with hydrazones of Girard's T and P reagents.

In this work synthesis, characterization and crystal structures of 1, Zn(II) complex ([ZnL1(NCS)2]), with (E)-1-(2-oxo-2-(2-(quinolin-2-ylmethylene)hydrazinyl)ethyl)pyridin-1-ium chloride (HL1Cl) and 2, Bi(III) complex ([BiHL2Cl4] × 1/2CH3OH), with (E)-N,N,N-trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride (HL2Cl), have been reported. Zn(II) complex possesses a distorted trigonal bipyramidal geometry while surroundings around Bi(III) ion are extended pentagonal bipyramidal. Antimicrobial activity, brine shrimp assay and DPPH radical scavenging activity of both complexes, including previously synthesized complexes with HL2Cl ligand (Zn(II) and Ni(II)) and complexes with (E)-N,N,N-trimethyl-2-oxo-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride (HL3Cl) (Zn(II), Cu(II), Cd(II), Co(II), Fe(III), Ni(II)), were evaluated. For the most active complexes, cytotoxic activity against five malignant cancer cell lines (HeLa, A375, MCF7, PC-3 and A549) and normal cell line HaCaT, as well as generation of reactive oxygen species (ROS), was tested.

Deciphering the Supramolecular Organization of Multiple Guests Inside a Microporous MOF to Understand their Release Profile.

Metal-organic frameworks (MOFs) give the opportunity of confining guest molecules into their pores even by a post-synthetic protocol. PUM168 is a Zn-based MOF characterized by microporous cavities that allows the encapsulation of a significant number of guest molecules. The pores engineered with different binding sites show a remarkable guest affinity towards a series of natural essential oils components, such as eugenol, thymol and carvacrol, relevant for environmental applications. Exploiting single crystal X-ray diffraction, it was possible to step-wisely monitor the rather complex three-components guest exchange process involving dimethylformamide (DMF, the pristine solvent) and binary mixtures of the flavoring agents. A picture of the structural evolution of the DMF-to-guest replacement occurring inside the MOF crystal was reached by a detailed single-crystal-to-single-crystal monitoring. The relation of the supramolecular arrangement in the pores with selective guests release was then investigated as a function of time and temperature by static headspace GC-MS analysis.

Synthesis of Imidazolidin-2-ones and Imidazol-2-ones via Base-Catalyzed Intramolecular Hydroamidation of Propargylic Ureas under Ambient Conditions.

The first organo-catalyzed synthesis of imidazolidin-2-ones and imidazol-2-ones via intramolecular hydroamidation of propargylic ureas is reported. The phosphazene base BEMP turned out to be the most active organo-catalyst compared with guanidine and amidine bases. Excellent chemo- and regioselectivities to five-membered cyclic ureas have been achieved under ambient conditions, with a wide substrate scope and exceptionally short reaction times (down to 1 min). A base-mediated isomerization step to an allenamide intermediate is the most feasible reaction pathway to give imidazol-2-ones, as suggested by DFT studies.

Stepwise Evolution of Molecular Nanoaggregates Inside the Pores of a Highly Flexible Metal-Organic Framework.

The crystalline sponge method (CSM) is primarily used for structural determination by single-crystal X-ray diffraction of a single analyte encapsulated inside a porous MOF. As the host-guest systems often show severe disorder, reliable crystallographic determination is demanding; thus the dynamics of the guest entering and the formation of nanoconfined molecular aggregates has not been in the spotlight. Now, the concept is investigated of the CSM for monitoring the structural evolution of nanoconfined supramolecular aggregates of eugenol guests with displacement of DMF inside the cavities of the flexible MOF, PUM168. The interpretation of the electron density provides a series of unique detailed snapshots depicting the supramolecular guest aggregation, thus showing the tight interplay between the host flexible skeleton and the molecular guests through the DMF-to-eugenol exchange process.

Comprehensive sequential genetic analysis delineating frequency, patterns, and prognostic impact of genomic dynamics in a real-world cohort of patients with lower-risk MDS.

The acquisition of subsequent genetic lesions (clonal evolution, CE) and/or the expansion of existing clones (CEXP) contributes to clonal dynamics (CD) in myelodysplastic syndromes (MDS). Although CD plays an important role in high-risk patients in disease progression and transformation into acute myeloid leukemia (AML), knowledge about CD in lower-risk MDS (LR-MDS) patients is limited due to lack of robust longitudinal data considering the long clinically stable courses of the disease. In this retrospective analysis, we delineate the frequency and the prognostic impact of CD in an unselected real-world cohort of LR-MDS patients. We screened 68 patients with a median follow-up of 40.5 months and a median of 7.5 (range: 2-22) timepoints for CE and CEXP detected by chromosomal banding analysis, fluorescence in situ hybridization, sequencing, and molecular karyotyping. In 30/68 patients, 47 CE events and a CD rate of 1 event per 4 years were documented. Of note, patients with at least 1 CE event had an increased probability for subsequent treatment. Unexpectedly, CE did not correlate with inferior outcomes, which could be reasonably explained by CD detection triggering the subsequent start of a disease-modifying therapy.

Fatal Progression of Mutated TP53-Associated Clonal Hematopoiesis following Anti-CD19 CAR-T Cell Therapy.

We present the case of a 64-year-old man diagnosed with large B-cell lymphoma who relapsed twice after standard-of-care therapy. Due to persisting cytopenia, Next generation sequencing analysis was performed, revealing a small TP53-mutated clone. As a third-line therapy, the patient was treated with CAR-T cells, which resulted in complete remission. However, this treatment also led to the expansion of the TP53-mutated clone and therapy-related myelodysplasia with a complex aberrant karyotype. This case may serve as a paradigmatic example of clonal hematopoietic progression in a patient undergoing CAR-T cell therapy, especially in the context of a TP53-mutated clone.

CAR-T Cell Therapy Shows Similar Efficacy and Toxicity in Patients With DLBCL Regardless of CNS Involvement.

Efficacy and toxicity of chimeric antigen receptor T (CAR-T) cell therapy in relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) with central nervous system (CNS) involvement remain understudied. Here we analyzed the outcomes of CAR-T cell therapy in r/r DLBCL patients with CNS involvement and compared them with patients without CNS disease. Retrospective and monocentric comparative analysis of patient cohort with r/r DLBCL treated with CAR-T cell therapy: 15 patients with CNS versus 65 patients without CNS involvement. Overall response rates (80% versus 80%; P = 1.0), progression-free survival (P = 0.157), and overall survival (P = 0.393) were comparable for both cohorts. The frequency of cytokine release syndrome was comparable in the CNS and non-CNS cohorts; 93% versus 80%; P = 1.0. Numerically, immune effector-cell-associated neurotoxicity syndrome (all grades) was more frequent in patients with CNS manifestation (53% versus 29%; P = 0.063), although no grade 4 events were documented. Our study suggests that CAR-T cell therapy is effective and feasible in patients with r/r DLBCL and CNS manifestation.

On the Mechanism of Cocrystal Mechanochemical Reaction via Low Melting Eutectic: A Time-Resolved In Situ Monitoring Investigation.

Mechanochemistry has become a sustainable and attractive cost-effective synthetic technique, largely used within the frame of crystal engineering. Cocrystals, namely, crystalline compounds made of different chemical entities within the same crystal structure, are typically synthesized in bulk via mechanochemistry; however, whereas the macroscopic aspects of grinding are becoming clear, the fundamental principles that underlie mechanochemical cocrystallization at the microscopic level remain poorly understood. Time-resolved in situ (TRIS) monitoring approaches have opened the door to exceptional detail regarding mechanochemical reactions. We here report a clear example of cocrystallization between two solid coformers that proceeds through the formation of a metastable low melting binary eutectic phase. The overall cocrystallization process has been monitored by time-resolved in situ (TRIS) synchrotron X-ray powder diffraction with a customized ball milling setup, currently available at µSpot beamline at BESSY-II, Helmholtz-Zentrum Berlin. The binary system and the low melting eutectic phase were further characterized via DSC, HSM, and VT-XRPD.

Overcoming the Drawbacks of Sulpiride by Means of New Crystal Forms.

This study aims at developing new multicomponent crystal forms of sulpiride, an antipsychotic drug. The main goal was to improve its solubility since it belongs to class IV of the BCS. Nine new adducts were obtained by combining the active pharmaceutical ingredient with acid coformers: a salt cocrystal and eight molecular salts. In addition, three novel co-drugs, of which two are molecular salts and one is a cocrystal, were also achieved. All samples were characterized in the solid state by complementary techniques (i.e., infrared spectroscopy, powder X-ray diffraction and solid-state NMR). For systems for which it was possible to obtain good-quality single crystals, the structure was solved by single crystal X-ray diffraction (SCXRD). SCXRD combined with solid-state NMR were used to evaluate the ionic or neutral character of the adducts. In vitro dissolution tests of the new crystal forms were performed and all the adducts display remarkable dissolution properties with respect to pure sulpiride.

Metal-organic framework-based magnetic dispersive micro-solid-phase extraction for the gas chromatography-mass spectrometry determination of polycyclic aromatic compounds in water samples.

A magnetic hybrid material based on the use of the mixed-ligand Metal-Organic Framework (MOF) PUM198 is proposed for the magnetic dispersive micro solid-phase extraction (MD-µSPE) of the 16 polycyclic aromatic hydrocarbons (PAHs) included in the US-EPA priority pollutants list. PUM198 is a thermally robust MOF characterized by a doubly interpenetrated microporous framework in which Zn2+ ions and carboxylate groups define 2D planes that are pillared by a bis-pyridine-bis amide ligand containing a biphenyl scaffold. PUM198 revealed to be ideal to adsorb PAHs efficiently through non-covalent interactions. A Plackett-Burman Design followed by a Central Composite Design and the multicriteria method of the desirability functions were applied to find the optimal conditions for the extraction of the investigated PAHs, resulting in a reduced solvent consumption, i.e., 50 µL of solvent per extraction for 5 mL of sample, approximatively 3-20 times lower than those reported in previous studies, thus satisfying the principles of green analytical chemistry. Method validation proved the reliability of the method for the determination of PAHs at trace level, obtaining detection limits in the 6.7-27 ng/L range, good precision with RSDs% lower than 19% and recovery rates in the 99 (±13)-126 (±8)% range near the quantitation limit. Finally, the applicability of the method was demonstrated by analyzing underground water samples taken from contaminated sites.

Comprehensive Laboratory Diagnostic Workup for Patients with Suspected Intraocular Lymphoma including Flow Cytometry, Molecular Genetics and Cytopathology.

BACKGROUND: Intraocular lymphoma (IOL) presents a real challenge in daily diagnostics. Cyto- and/or histopathology of vitreous body represent the diagnostic cornerstones. Yet, false negative results remain common. Therefore, we analyzed the diagnostic significance of flow cytometry (FC) within the workup algorithm of IOL and compared its sensitivity with the results obtained from routine cytopathology and molecular genetics; Methods: Seven patients undergoing vitrectomy due to suspected IOL were investigated by FC and parallel cytopathology and, if available, digital droplet PCR (ddPCR) for MYD88 L265P; Results: Four out of seven patients were finally diagnosed with IOL. Among the IOL patients, cytopathology confirmed the presence of lymphoma cells in only two cases. In contrast, FC was positive for IOL in all four cases, and FC additionally confirmed the lack of IOL in the remaining patients. In IOL patients diagnosed by FC and with available ddPCR, the diagnosis of IOL was confirmed by the presence of the MYD88 L265P mutation in all three patients; Conclusions: The combination with FC was superior to cytopathology alone in the diagnostic work-up of IOL, and it showed an excellent correlation with ddPCR results. A comprehensive diagnostic panel consisting of cytopathology, FC and molecular genetics should be considered for the work-up of suspected IOL.

Mechanochemical Preparation of Dipyridyl-Naphthalenediimide Cocrystals: Relative Role of Halogen-Bond and π-π Interactions.

Naphthalenediimide derivates are a class of π-conjugated molecules largely investigated in the literature and used as building blocks for metal-organic frameworks or coformers for hydrogen-bond-based cocrystals. However, their tendency to establish halogen-bond interactions remains unexplored. By using a crystalline engineering approach, we report here four new cocrystals with N,N'-di(4-pyrydyl)-naphthalene-1,4,5,8-tetracarboxidiimide and diiodo-substituted coformers, easily obtained via a mechanochemical protocol. Cocrystals were characterized via NMR, electron ionization mass spectrometry, thermogravimetric analysis, powder X-ray diffraction, and single-crystal X-ray diffraction. Crystallographic structures were then finely examined and correlated with energy framework calculations to understand the relative contribution of halogen-bond and π-π interactions toward framework stabilization.