Blood Cancer Journal

Acute myeloid leukemia (AML), the most common hematologic malignancy, generally has a poor prognosis. Despite initial favorable responses to the BCL2 inhibitor venetoclax (VEN), remission is transient, and AML is eventually fatal. Resistance to VEN is primarily due to the overexpression of anti-apoptotic proteins, including MCL-1, BCL2L1 (BCL-XL), and BCL2A1. Casein kinase II (CK2) is a serine-threonine kinase and a known suppressor of apoptosis. We and others have reported that protein kinase CK2 activity is high in leukemic stem cells (LSCs) and associated with resistance to chemotherapy. We have shown that the selective CK2 inhibitor, CX-4945, suppresses BCL-XL and has a significant anti-tumor effect in AML preclinical models. CK2 expression and activity are high in venetoclax-resistant AML (VR-AML) cell lines. Genetic and pharmacological inhibition of CK2 significantly altered VR-AML gene signature, decreased MCL-1 protein level, increased BH3 priming and sensitized VR-AML cells to apoptosis. More importantly, CX-4945 selectively targeted LSCs (CD34+CD38-) and chemoresistant (CD123+CD47+) subpopulation in VR-AML. CX-4945 combined with VEN decreased leukemia burden and prolonged the survival of VR-AML cell line-derived and patient-derived xenografts compared to either drug alone. The combinatorial treatment was well tolerated in mice without additional myelosuppression or organ toxicity. CX-4945 (silmitasertib) is being tested in several early-phase clinical trials against adult and pediatric cancers. These preclinical results support the use of CX-4945 in combination with VEN to overcome resistance to apoptosis and re-sensitize VR-AML to chemotherapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/696284v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6a902forg.highwire.dtl.DTLVardef@2028f5org.highwire.dtl.DTLVardef@160fd4dorg.highwire.dtl.DTLVardef@95da53_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite a record number of clinical studies investigating various anti-cancer drugs, the 5-year survival rate for multiple myeloma (MM) patients in the United States is only 55%, and nearly all patients relapse. Poor patient outcomes demonstrate that myeloma cells are "born to survive," which means they can adapt and evolve following treatment. As a result, new therapeutic approaches to combat this survival mechanism and target treatment-resistant malignant cells are required. Mcl-1, an anti-apoptotic protein, is required for the development of MM and resistance to therapy. This study looks at the possibility of KS18, a Mcl-1 inhibitor derived from pyoluteorin, to treat resistant MM. We show that KS18 inhibits Mcl-1 selectively and promotes post-translational modifications, resulting in UPS-dependent Mcl-1 degradation. Our findings show that KS18-induced Mcl-1 degradation results in caspase-dependent apoptosis. Importantly, KS18 triggered apoptosis in MM patient samples and bortezomib-resistant cells, synergizing with venetoclax to boost apoptosis. Furthermore, KS18 inhibits colony formation in bortezomib-resistant cells. KS18 treated NSG mice displayed significant tumor shrinkage without significant toxicity after four weeks of therapy with a single acceptable dose each week, indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance. Key pointsO_LIGiven that KS18 is a robust Mcl-1 inhibitor that targets Mcl-1 efficiently, it has the potential to be a novel treatment for multiple myeloma. C_LIO_LIKS18 has shown promise in re-sensitizing myeloma cells to chemotherapy as well as in overcoming resistance to bortezomib, venetoclax, and ABT-737. C_LI

BackgroundHematological malignancies include diverse cancers related to immune system cells and blood-forming tissue. Treatment resistance and relapse are common despite the introduction of novel therapeutics. Following the development of the PET probe [89Zr]Zr-DFO-isatuximab, targeting the overexpressed CD38 receptor, we developed [225Ac]Ac-Macropa-isatuximab for targeted alpha therapy in murine models of human multiple myeloma (MM) and lymphoma. MethodsIn vitro studies were performed using the CD38 expressing MM1.S (human MM) and Daudi (human lymphoma). Radiopharmaceutical therapy studies were performed in female NSG mice, and the disseminated disease models were established by intravenous injection of luciferase-transfected cells. Mice were randomized (n = 10 mice per cohort) and received a high activity (5.5 kBq) or low activity (2.7 kBq) of [225Ac]Ac-Macropa-Isatuximab in single or multiple cycles. Controls receiving untargeted [225Ac]Ac-Macropa-IgG or saline were included. Tumor burden was measured by bioluminescence imaging (BLI). Weight loss greater than 20% or leg paralysis were used as endpoints. Results[225Ac]Ac-Macropa-Isatuximab was obtained with high radiochemical yield and purity (>95%). It displayed high immunoreactivity and excellent stability in human serum over 10 days. The therapeutic effect of [225Ac]Ac-mcp-Isatuximab was significant for all the treatment cohorts compared to the saline control after administration of the first cycle. Low activities were better tolerated in general, resulting in extended median survivals. The most successful regimen in the MM model was the dual cycle of low activity, with a median survival of 60 days (P < 0.001). In the lymphoma model, the single high activity of 5.5 kBq and four low activity of 2.7 kBq resulted in similar median survivals of 143 days (P < 0.0001) and 147 days (P < 0.0001). These regimens resulted in complete responses with no detectable cancer cells in some cases. Remarkably, no significant kidney damage was observed in any of the models. Conclusion[225Ac]Ac-Macropa-Isatuximab selectively targets CD38, reducing tumor growth and, in some cases, tumor eradication, especially when applied in multiple cycles. Importantly, it has not been associated with significant toxicities at levels of administered activity required for therapeutic benefit, indicating its promise as a therapeutic option.

Diffuse large B-cell lymphoma (DLBCL) represents 30-40% of non-Hodgkin lymphoma cases and is curable in >60% of patients; however, approximately one-third ultimately relapse. Although prior studies in normal B cells and lymphoma models implicate p73 in B-cell lymphomagenesis, the functional role of individual p73 isoforms in DLBCL remains poorly defined. TP73, a TP53 family member located on chromosome 1p36, encodes both transcriptionally active (TAp73) and dominant-negative ({Delta}Np73) isoforms that differentially regulate apoptosis and proliferation. In this study, we characterized p73 locus alterations, isoform-specific expression patterns, and their biological relevance in DLBCL. Chromosomal analysis revealed disruption of the 1p36 locus--predominantly via heterozygous deletion--in 35% of patient samples, which significantly correlated with elevated {Delta}Np73 expression. Immunohistochemical profiling demonstrated a positive association between TAp73 and cleaved caspase-3, and between {Delta}Np73 and Ki-67. Conversely, TAp73 expression negatively correlated with the anti-apoptotic proteins Bcl-2 and Bcl-6. Functional studies in DLBCL cell lines further confirmed that TAp73 enhances sensitivity to serum deprivation and doxorubicin, whereas {Delta}Np73 overexpression promotes survival and chemoresistance. Together, these findings identify p73 isoform imbalance as a key contributor to DLBCL pathogenesis and therapeutic response, highlighting {Delta}Np73 as a potential biomarker of aggressive disease and treatment resistance, and TAp73 as a tumor-suppressive axis warranting further investigation. SummaryDiffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, yet relapse remains a major challenge. The p73 gene produces two key isoforms with opposing functions: TAp73, which promotes apoptosis, and {Delta}Np73, which inhibits cell death and supports tumor growth. In DLBCL samples, 1p36 chromosomal disruption occurred in 35% of cases and was associated with elevated {Delta}Np73. TAp73 expression correlated with apoptosis markers, whereas {Delta}Np73 correlated with proliferation. Functional studies showed TAp73 sensitizes DLBCL cells to stress and chemotherapy, while {Delta}Np73 enhances resistance. These findings highlight {Delta}Np73 as a potential biomarker and therapeutic target in DLBCL.

BackgroundNovel drugs are dynamically changing current treatment regimens for multiple myeloma (MM). Novel drugs have improved prognosis of MM patients in clinical studies but are expensive. Little is known about up-to-date real-world application and costs. MethodsWe performed a retrospective observational cohort analysis (cost-outcome description; 2012-2017) in a claims database of a major Swiss health insurance company which covers 14% of the Swiss population (Helsana Versicherungen AG).We used primary (MM diagnoses via ICD-10) and secondary features (prescribed MM-specific drugs) as inclusion criteria and defined a hierarchy of drug regimens to classify treatments as: 1) proteasome inhibitor (PI)-based regimen (e.g. bortezomib); 2) IMID-based regimen (e.g. lenalidomide); 3) chemotherapy (CHEMO)-based regimen (e.g. bendamustin); 4) monoclonal antibody (MAB)-based regimen (e.g. daratunumab). Direct medical costs of mandatory health insurance were analysed in 2017 Swiss Francs (CHF; third party payer perspective). ResultsOverall, we identified n=1054 prevalent MM patients (2012-2017) and n=378 incident MM patients (2015-2017; men: 47.1%; age group <=75 years: 48.7%). The number of prevalent patients per year increased over time (from n=314 in 2012 to n=645 in 2017). PI-based regimens were the most frequent first line approach for incident patients (76.0%), followed by IMID-based (21.9%) and CHEMO-based regimens (2.1%). Only four patients were treated with MAB-drugs. For later lines, IMID-based regimens were most often used (2nd line: 56.4%; 3rd line: 2 of 3 patients), followed by PI-based regimens (43.6% and 1 of 3 patients, respectively). 161 of 1054 prevalent MM patients (15.3%) were treated with autologous hematopoietic stem cell transplantation (HSCT), 4 patients with allogeneic HSCT. Average costs per patient per treatment line varied considerably (reliable data available from 2012 to 2014; mean duration of lines between 112 and 388 days): PI-based regimens: CHF 81352; IMID-based: CHF 73495; CHEMO-based: CHF 683. Mean daily costs under MM treatment stepwise increased from CHF 209 in 2012 to CHF 254 in 2017 (relative increase: 21.5%). Annual direct medical costs in Switzerland for seven novel MM drugs were extrapolated to be 60.1 Mio CHF in 2012 and 118.6 Mio CHF in 2017 (relative increase: 97.3%), corresponding to mean annual outpatient MM drug costs per patient of CHF 28000 in 2017. Annual death rates decreased systematically from 18.6% in 2012 to 15.5% in 2017 (p for trend: 0.03). No statistically significant difference in death rates emerged for 2017 compared with 2012 (risk ratio: 0.83; 95%-CI: 0.63 to 1.10; absolute risk reduction: 3.1%). ConclusionsCurrent treatment patterns for MM patients in Switzerland show variation concerning applied drug regimens as well as costs. An increasing prevalent population of MM patients in combination with increasing costs per day under treatment lead to a substantial and growing budget impact for the Swiss social insurance system.

Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.

1)Diffuse Large B-Cell Lymphoma (DLBCL) are the most common form of non-Hodgkin lymphoma. Their molecular origin is heterogeneous and therefore treatments aimed at DLBCL must be adapted in function of the underlying molecular mechanisms driving cellular transformation. Constitutive activation of the protein kinases ERK1/2 is a hallmark of many B-cell malignancies. ERK1/2 activation which can occur downstream of the classical MAPK cascade via RAF or, in response to TLR stimulation, via the Tumor Promoting Locus 2 (TPL2) protein kinase. This pathway also relays signals from the MYD88 oncogenic mutant L265P, frequently found in hematologic malignancies. We report here that TPL2 participate to ERK1/2 activation downstream of BCR in a DLBCL cell line (OCI-Ly2). Moreover, we showed that a ERK2[Y316F] mutant increased c-Myc-luciferase reporter expression. We then investigated the impact of ERK1/2 inhibition on the proliferation of OCI-Ly2 cells. We found that blocking ERK1/2 MAPK signaling cascade using either MKK1/2 inhibitors (PD184352 and MEK162) or TPL2 inhibitor (Compound 1) was mainly cytostatic. Finally, we showed that while TPL2-MKK1/2 inhibition leads to cytostatic effect, Compound 1 has cytocidal effect at high concentrations, that is mediated via additional targets. Taken together, this study demonstrates the involvement of TPL2 in oncogenic signaling of DLBCL and supports the idea that combination therapy targeting multiple molecular pathways linked to cellular transformation is a superior avenue for future therapies.

TP53 mutations, including missense and inactivating (frameshift, splice site, and nonsense) mutations, occur in approximately 10% of myeloid neoplasms and confer adverse outcomes. Classification of myeloid neoplasms by both the World Health Organization and the International Consensus Classification standards now recognize the prognostic and therapeutic importance of early detection of TP53 mutations. p53 immunohistochemistry (IHC) is a simple and rapid method commonly used to detect p53 mutations. More recently, sequencing via targeted panels has also seen increased use. While highly accurate, sequencing is resource intensive and not universally available. IHC represents a more accessible option for mutation detection, however previous studies have demonstrated variable accuracy, especially for inactivating TP53 mutations. Using 134 bone marrow core samples of acute myeloid leukemia (AML) evaluated for TP53 mutation by a sequencing panel, we assessed the concordance of p53 IHC with sequencing as well as the inter-rater reliability for IHC intensity and percent positivity. Consistent with previous studies, we found that p53 IHC was strongly specific and modestly sensitive for missense mutations, and that overall performance improved with dedicated hematopathology training. We also found that IHC performed poorly for inactivating mutations and was even variable between cases harboring identical amino acid changes. Low predicted transcriptional activity of TP53 missense mutations correlated with a mutant pattern of IHC staining. The status of the second allele in missense mutations and variant allele fraction also affected the accuracy of p53 IHC as a surrogate for TP53 allele status. AMLs expressing p53 mutations that were predicted to have low transcriptional activity correlated with reduced overall survival. Our results demonstrate limited practical utility of p53 immunohistochemistry for accurate evaluation of TP53 mutation status due to multifactorial confounders.

Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive proliferation of myeloid lineages. Accurate classification and appropriate management of MPNs requires integration of clinical, morphological and genetic findings. Despite major advances in understanding the molecular and genetic basis, morphological assessment of the bone marrow trephine (BMT) remains paramount in differentiating between MPN subtypes and reactive conditions. However, morphological assessment is heavily constrained by a reliance on subjective, qualitative and poorly reproducible criteria. To address this, we have developed a machine-learning strategy for the automated identification and quantitative analysis of megakaryocyte morphology using clinical BMT samples. Using a sample cohort of recently diagnosed or established ET (n = 48) and reactive control cases (n = 42) we demonstrated a high predictive accuracy (AUC = 0.95) of automated tissue ET diagnosis based upon these specific megakaryocyte phenotypes. These separate morphological phenotypes showed evidence of specific genotype associations, which offers promise that an automated cell phenotyping approach may be of clinical diagnostic utility as an adjunct to standard genetic and molecular tests. This has great potential to assist in the routine assessment of newly diagnosed or suspected MPN patients and those undergoing treatment / clinical follow-up. The extraction of quantitative morphological data from BMT sections will also have value in the assessment of new therapeutic strategies directed towards the bone marrow microenvironment and can provide clinicians and researchers with objective, quantitative data without significant demands upon current routine specimen workflows.

TP53 mutation predicts adverse prognosis in many cancers, including myeloid neoplasms, but the mechanisms by which specific mutations impact disease biology, and whether they differ between disease categories, remain unknown. We analyzed TP53 mutations in four myeloid neoplasm subtypes (MDS, AML, AML with myelodysplasia-related changes (AML-MRC), and therapy-related acute myeloid leukemia (tAML)), and identified differences in mutation types, spectrum, and hotspots between disease categories and compared to solid tumors. Missense mutations in the DNA-binding domain were most common across all categories, whereas inactivating mutations and mutations outside the DNA binding domain were more common in AML-MRC compared to MDS. TP53 mutations in MDS were more likely to retain transcriptional activity, and co-mutation profiles were distinct between disease categories and mutation types. Our findings suggest that mutated TP53 contributes to initiation and progression of neoplasia via distinct mechanisms, and support the utility of specific identification of TP53 mutations in myeloid malignancies. Statement of SignificanceThe distribution and functional consequences of TP53 mutations differ between hematologic malignancies and solid tumors, and, among myeloid neoplasms, between myelodysplastic syndrome and acute leukemia. These findings suggest distinct biological mechanisms for mutated p53 in hematologic malignancies, specifically in initiation and progression of myeloid neoplasia, that warrant further investigation.

Measurable residual disease (MRD), defined as the population of cancer cells which persists following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia (AML) and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones and normal precursors remains a challenge with current MRD tools. Herein, we developed a single cell (sc) MRD assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated scDNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/pre-leukemic and leukemic cells surviving AML therapy. Statement of significanceScMRD assay integrates mutation and immunophenotype at single cell resolution and therefore distinguishes clonal hematopoiesis/preleukemic vs. leukemic clones. This study serves as a framework for identifying high-risk MRD clones and improving our understanding of both the molecular drivers and vulnerabilities of therapy resistant AML clones.

BackgroundChronic Eosinophilic Leukemia, Not Otherwise Specified (CEL-NOS), a rare and intricate hematological disorder characterized by uncontrolled eosinophilic proliferation, presents clinical challenges owing to its infrequency. This study aimed to investigate the epidemiology and develop a prognostic nomogram for CEL-NOS patients. MethodsUtilizing the Surveillance, Epidemiology and End Results (SEER) database, CEL-NOS cases diagnosed between 2001 and 2020 were analyzed for incidence rates, clinical profiles, and survival outcomes. Patients were randomly divided into training and validation cohorts (7:3 ratio). LASSO regression analysis and Cox regression analysis were performed to screen the prognostic factors for overall survival. A nomogram was then constructed and validated to predict the 3- and 5-year overall survival probability of CEL-NOS patients by incorporating these factors. ResultsThe incidence rate of CEL-NOS was very low, with an average of 0.033 per 100,000 person-years from 2001 to 2020. The incidence rate significantly increased with age and was higher in males than females. The mean age at diagnosis was 57 years. Prognostic analysis identified advanced age, specific marital statuses, and secondary CEL-NOS as independent and adverse predictors of overall survival (OS). To facilitate personalized prognostication, a nomogram was developed incorporating these factors, demonstrating good calibration and discrimination. Risk stratification using the nomogram effectively differentiated patients into low- and high-risk groups. ConclusionsThis study enhances our understanding of CEL-NOS, offering novel insights into its epidemiology, demographics, and prognostic determinants, while providing a possible prognostication tool for clinical use. However, further research is warranted to elucidate molecular mechanisms and optimize therapeutic strategies for CEL-NOS.

IntroductionAcute lymphoblastic leukemia (ALL) is the most common childhood cancer, with rising global incidence. The prognosis for patients with relapsed or refractory B-cell ALL (r/r B-ALL) remains poor, necessitating novel therapies. Chimeric Antigen Receptor T-cell (CAR T-cell) therapy has shown promise in treating r/r B-ALL, offering significant improvements in remission rates. MethodsA comprehensive literature search was conducted across PubMed, Embase, and Cochrane Library for studies evaluating CAR T-cell therapy in r/r B-ALL. Randomized controlled trials, cohort, and case-control studies were included, focusing on efficacy and safety outcomes. Data were extracted and pooled using random-effects models. The risk of bias was assessed with the New Ottawa Scale. ResultsA total of 32 studies were included, involving 1,55,365 patients. The pooled relapse rate was 0.39 (95% CI: 0.29-0.49), with no significant difference between CD19 and CD22 CAR T-cell therapies (p = 0.88). Co-stimulatory agents like 4-1BB showed the most favorable relapse rate of 0.38 (95% CI: 0.27-0.49). The overall Cytokine Release Syndrome (CRS) rate was 0.63 (95% CI: 0.53-0.73), and neurotoxicity occurred at a rate of 0.32 (95% CI: 0.24-0.41). ConclusionCAR T-cell therapy is effective in treating r/r B-ALL, with high remission rates and manageable adverse events. The choice of co-stimulatory agent and antigen target influences relapse outcomes. Further research is needed to refine CAR T-cell constructs and optimize patient-specific treatments.

Acute myeloid leukaemia (AML) is a haematopoietic malignancy comprising different genetic subtypes with a common hallmark of differentiation arrest. In abnormal haematopoiesis, overcoming the differentiation blockade has emerged as an attractive therapeutic strategy. In a screen with genetically distinct AML cell lines, histone deacetylase inhibitors (HDACis) were observed to cause an upregulation in the expression of CD11b, a myeloid differentiation marker. These caused changes in cell morphology, block in proliferation, and cell cycle arrest at the G1 phase. To gain insights onto the mechanism of these compounds, we planned to prepare inactive probes devoid of the zinc binding motif. However, these compounds were unexpectedly still able to initiate differentiation, albeit through a distinct target and via a G2 arrest. Subsequent RNA sequencing studies supported the differentiation phenotype for the HDACis and highlighted the role of cell cycle regulatory kinases for the effect observed in the probe molecules. We then showed that these inhibit Aurora A and GSK3 kinases, suggesting their potential as therapeutic targets for differentiation therapy in AML. Our work supports the importance of properly validating inactive tool compounds and their potential to identify novel targets.

Despite promising anti-leukemic activity of MCL-1 inhibitors in preclinical studies of acute myeloid leukemia (AML), their progress through clinical evaluation has in part been challenged by limited knowledge of patient subgroups suitable for treatment. To stratify patients with AML for MCL-1 inhibitor-based treatment, we evaluated the sensitivity of 42 primary AML samples to MCL-1 inhibitor MIK665 (S64315) and contrasted their molecular profiles. We observed that MIK665 sensitive samples had a more differentiated phenotype, whereas resistant samples displayed higher levels of ABCB1 (MDR1) and the anti-apoptotic protein BCL-XL. Further evaluation revealed that ABCB1 expression has good predictive performance in identifying MIK665 primary resistant samples. To induce sensitivity, we treated MIK665-resistant samples with ABCB1 inhibitor elacridar, BCL-XL inhibitor A1331852, or BCL-2 inhibitor venetoclax in combination with MIK665. While combinations with elacridar and A1331852 were not effective, the combination of MIK665 and venetoclax effectively eliminated AML blasts compared to either of the agents alone. Additionally, the combination restored sensitivity of samples with primary venetoclax resistance. Overall, this study indicates that elevated ABCB1 expression is a potential predictor of resistance to MIK665 in AML, and that a combination of MIK665 with venetoclax may be effective for overcoming resistance to either MCL-1 or BCL-2 inhibition.

Chronic myelomonocytic leukemia (CMML) is a clonal myelodysplastic/myeloproliferative neoplasm characterized by persistent monocytosis and heterogeneous risk of progression to acute leukemia. Mutations within the RAS/MAPK signaling pathway, particularly involving KRAS, are linked to a proliferative disease phenotype and adverse prognosis. We report the first Colombian CMML case harboring two concurrent activating KRAS mutations (p.G12S and p.G13D). Both variants were detected at variant allele frequencies of 17% and 21% in a female patient in her late 50s presenting with marked leukocytosis, anemia, and thrombocytopenia. The coexistence of these mutations suggests synergistic hyperactivation of the RAS/MAPK pathway, likely driving clinical aggressiveness and therapeutic resistance. This case delineates a rare molecular subgroup within CMML and highlights the critical role of comprehensive genomic profiling to improve prognostic accuracy and inform precision medicine approaches.

The 26S proteasome is an essential regulator of protein homeostasis and a clinically validated therapeutic target in multiple myeloma (MM). Rapaprotin, a novel macrocycle identified from a rapamycin-inspired rapafucin library, disrupts 26S proteasome function by inducing disassembly of the 19S regulatory particle in the 26S proteasome, leading to apoptosis in MM cells. Its bioactivation requires prolyl endopeptidase (PREP)-mediated cleavage to generate Rapaprotin-L, a negatively charged, linear metabolite with potent proteasome-disassembly activity. Using the PRISM cancer cell line profiling platform, we identified high P-glycoprotein (P-gp/ABCB1) expression as a major determinant of Rapaprotin resistance in solid tumor cell lines. Efflux assays confirmed Rapaprotin-L, but not its parent Rapaprotin, as a high-affinity P-gp substrate. Co-treatment with the third-generation P-gp inhibitor tariquidar restored the intracellular accumulation of Rapaprotin-L, reinstating proteasome inhibition and consequent apoptosis of Rapaprotin-resistant colorectal cancer cell lines. Strong synergy between Rapaprotin and tariquidar was observed in a 3D spheroid model. These results establish P-gp as a key mediator of resistance to Rapaprotin and identify a rare example of a negatively charged Rapaprotin-L as a P-gp substrate. Together, these findings expand the potential therapeutic scope of Rapaprotin beyond hematologic malignancies to a broader range of solid tumors.

RationaleMultiple Myeloma (MM) stands as the second most common hematological malignancy characterized by the accumulation of monoclonal plasmocytes within the bone marrow. Despite the introduction of proteasome inhibitors, immunomodulatory agents and CD38-targeting antibodies which have extended survival rates, the disease remains incurable for most patients due to the emergence of resistant clones and frequent relapses. The efficacy of the proteasome inhibitor bortezomib (BTZ) in MM treatment underscores the critical role of the ubiquitin proteasome system (UPS) in this cancer. Deubiquitinases (DUBs), a class of enzymes governing the stability, interactions or localization of cellular proteins by removing ubiquitin modifications, have emerged as promising therapeutic targets across various cancers, including MM. MethodsThrough an exhaustive loss-of-function approach, we have identified for the first time USP39 DUB as a pivotal survival determinant for MM cells. ResultsOur analysis reveals a direct correlation between heightened USP39 mRNA levels and shorter survival in MM patients. Additionally, robust USP39 protein expression is observed in MM patient plasmocytes compared to healthy counterparts. Knockdown of Usp39 not only impedes clonogenic capabilities, but also induces apoptosis, triggers cell cycle arrest and overcomes BTZ resistance. Complementary gain-of-function assays, further elucidate how USP39, by stabilizing the transcription factor ZEB1, enhances the trans-migratory potential of MM cells. ConclusionsIn summary, our findings underscores the pivotal role of the deubiquitinase USP39, suggesting that targeting the USP39/ZEB1 axis hold promise as a prospective diagnostic marker and therapeutic target in MM.

BackgroundPatients with relapsed/refractory multiple myeloma (RRMM) and high-risk genetic abnormalities such as del(17p) and TP53 mutation have poor response to standard therapies and shorter survival compared to patients without these aberrations. Here, we investigated the activity and mechanism of action of peptide-drug conjugate melphalan flufenamide (melflufen) in TP53 wild type (TP53wt) and mutant (TP53mut) myeloma models and assessed the efficacy of melflufen in patients with del(17p) and/or TP53 mutation. MethodsWe evaluated melflufen activity ex vivo in 24 myeloma bone marrow (BM) samples and explored indicators of response from single cell RNA sequencing (scRNAseq) profiles. The efficacy of melflufen vs. control treatments was further investigated in TP53-/- and parental TP53wt myeloma cell lines. DNA damage, apoptosis kinetics, mitochondrial function, plus transcriptomic and metabolic data were analyzed to understand the mechanisms responsible for melflufen activity in the absence of p53. Patient outcome data from the OCEAN phase III clinical trial (NCT03151811), which investigated the clinical activity of melflufen in RRMM, were statistically analyzed to assess the impact of del(17p) and TP53 mutation on clinical response. ResultsBM plasma cell (PC) response to melflufen was independent of TP53 mutation status, with melflufen active in del(17p), TP53mut, and TP53wt samples. Differential analysis of scRNAseq data demonstrated that melflufen sensitive PCs had lower expression of p53 target genes and higher expression of genes associated with DNA damage repair and cell cycle checkpoints. Analysis of TP53-/- and TP53wt cell lines showed superior efficacy of melflufen in comparison to melphalan or cyclophosphamide. In the presence and absence of functional p53, melflufen robustly induced apoptosis, DNA damage, and mitochondrial dysfunction. In TP53-/- cells, melflufen treatment led to distinct changes in expression of genes associated with cell cycle checkpoint and apoptosis, which were not observed with melphalan treatment. Notably, post-hoc analysis of the OCEAN trial del(17p) patient population demonstrated favorable progression free survival in the del(17p) subgroup treated with melflufen plus dexamethasone compared to the pomalidomide plus dexamethasone arm. ConclusionsOur insights into the molecular mechanisms of melflufen activity in TP53mut myeloma support its clinical efficacy and application in the del(17p) and TP53mut patient population. Trial registrationNCT03151811, registration 2017-05-09.

BackgroundMultiple myeloma (MM) is characterized by abnormal plasma cell proliferation in the bone marrow, leading to symptoms like osteolytic lesions, anemia, hypercalcemia, and elevated serum creatinine. RNA-sequencing-based prognostic indicators for MM have shown promise in stratifying risk and assessing first-line treatment options. This study uses machine learning techniques and leverages RNA-sequencing, clinical, and biochemical data from the Multiple Myeloma Research Foundation (MMRF) CoMMpass cohort to predict patient prognosis. MethodsRNAseq data of 60,623 genes from bone marrow samples of 708 MM patients were pre-processed for batch effect correction and split into training (70%) and testing (30%) sets. Feature selection involved MAD, mRMR, and iterative permutation importance filtering for predicting PFS and OS. Machine learning survival models like Random Survival Forest (RSF), Gradient Boosted (GB), and Component-wise Gradient Boosted (CGB) were developed and optimized. Performance was evaluated using C-index and integrated Brier score (IBS). ResultsThe RSF and GB models showed the highest performance for predicting progression-free survival (PFS) and overall survival (OS) on the testing dataset. Significant features for PFS included stem cell transplant status, serum {beta}2-microglobulin levels, germline mutational status, and expression of C12orf75 and ENSG00000256006. For OS, stem cell transplant status, age, serum {beta}2-microglobulin levels, germline mutational status, and expression of NUTM2B-AS1 and ENSG00000287022 were prominent. Gene ontology analyses confirmed the biological relevance of enriched pathways related to cell division, protein localization, and cancer. ConclusionIntegrating RNAseq and clinical data with advanced machine learning models presents a robust approach for predicting MM prognosis, highlighting gene expression programs, germline mutational status, and clinical markers as significant features. Future research should focus on independent validation to confirm findings and explore additional genomic data for enhanced prognostication.