Monica Facco

Background/Objectives: Identifying molecular liabilities and understanding disease heterogeneity are prerequisites for advancing therapies in T-cell prolymphocytic leukemia (T-PLL), a rare T-cell malignancy with a poor prognosis. Methods: RNA-seq profiling of T-PLL samples (n = 10) and the normal counterpart (n = 5) allowed us to report gene expression and pathway alterations in malignant cells, revealing that non-coding, antisense and circular RNA expression is profoundly altered in T-PLL. Results: T-PLL displayed activation of several oncogenic pathways, particularly PI3K/AKT/mTOR and Wnt, suppression of healthy T-cell activities and cell death escape. Tumor suppressor lncRNAs (NEAT1, MIAT and LUCAT1) with reduced expression and upregulated oncogenic pro-proliferative lncRNAs (FIRRE, TERC, XIST and PVT1) were identified. CircRNAs ectopically expressed in T-PLL included circSEMA4B and circSATB1, linked to the Wnt pathway, circFIRRE and oncogenic circPVT1 and circFKBP5. Focusing on five genes with validated recurrent oncogenic variants (STAT5B, JAK3, ATM, KMT2C, and ARID1A), we investigated genotype/phenotype relations. A multiple predictor linear model suggested potential links between driver variants and alterations in gene and circRNA expression, including association between STAT5B mutations and LTF upregulation, JAK3 lesions and increased PLXNA4 expression along with CCR4 suppression. Conclusions: Our transcriptomic profiling and genotype–phenotype association analysis identified specific genes, non-coding RNAs, pathways and candidate genotype-associated transcriptional signatures that warrant further investigation as potential targets for the development of new therapeutic approaches for this rare and heterogeneous malignancy.

The biological basis of the high clinical heterogeneity of T-LGL Leukemia (T-LGLL) is not completely understood and effective therapies for this disease are lacking. Through RNA-Sequencing of purified T-LGLs we reveal gene expression profiles and pathway dysregulations in the major patient subgroups, defined by CD8+ or CD4+ phenotype and STAT3 / STAT5B mutational status. Overall, T-LGLL patients exhibited a marked transcriptome dysregulation compared to controls. This was more pronounced in the most symptomatic CD8 + STAT3 -mutated patients, which emerged as a distinct biological entity, separated from the other disease subgroups. Particularly, CD8 + STAT3 -mutated cases displayed extensive down-regulation of genes, ultimately resulting in the de-repression of proliferation and cell cycle pathways. Among genes up-regulated in CD8 + STAT3 -mutated cases we found VCAM1 , the transcriptional repressor EZH2 and the p53-regulator MDM2 proto-oncogene, as well as the leukemogenesis-associated PVT1 up-regulation, representing the first report of a long-non-coding RNA alterations in leukemic T-LGLs. The impact of STAT5B mutations on T-LGLs transcriptome was more limited and the overexpression of the PIM1 serine/threonine kinase proto-oncogene was identified as one of the most relevant features of STAT5B -mutated CD4 + T-LGLL. This study significantly advances our understanding of T-LGLL pathogenesis, uncovering new oncogenic mechanisms within the distinct molecular subtypes of the disease.

Finding an effective treatment for T-PLL patients remains a significant challenge. Alemtuzumab, currently the gold standard, is insufficient in managing the aggressiveness of the disease in the long term. Consequently, numerous efforts are underway to address this unmet clinical need. The rarity of the disease limits the ability to conduct robust clinical trials, making in silico, ex vivo, and in vivo drug screenings essential for designing new therapeutic strategies for T-PLL. We conducted a drug repurposing analysis based on T-PLL gene expression data and identified proteasome inhibitors (PIs) as a promising new class of compounds capable of reversing the T-PLL phenotype. Treatment of ex vivo T-PLL cells with Bortezomib and Carfilzomib, two PI compounds, supported this hypothesis by demonstrating increased apoptosis in leukemic cells. The current lack of a suitable in vitro model for the study of T-PLL prompted us to perform similar experiments in the SUP-T11 cell line, validating its potential by showing an increased apoptotic rate. Taken together, these findings open new avenues for investigating the molecular mechanisms underlying the efficacy of PI in T-PLL and expand the spectrum of potential therapeutic strategies for this highly aggressive disease.

Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; p = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; p = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.

The search for molecules to be targeted that are involved in apoptosis resistance/increased survival and pathogenesis of onco-hematological malignancies is ongoing since these diseases are still not completely understood. Over the years, a good candidate has been identified in the Heat Shock Protein of 70kDa (HSP70), a molecule defined as “the most cytoprotective protein ever been described”. HSP70 is induced in response to a wide variety of physiological and environmental insults, allowing cells to survive lethal conditions. This molecular chaperone has been detected and studied in almost all the onco-hematological diseases and is also correlated to poor prognosis and resistance to therapy. In this review, we give an overview of the discoveries that have led us to consider HSP70 as a therapeutic target for mono- or combination-therapies in acute and chronic leukemias, multiple myeloma and different types of lymphomas. In this excursus, we will also consider HSP70 partners, such as its transcription factor HSF1 or its co-chaperones whose druggability could indirectly affect HSP70. Finally, we will try to answer the question asked in the title of this review considering that, despite the effort made by research in this field, HSP70 inhibitors never reached the clinic.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRβ sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.

The Btk inhibitor ibrutinib has significantly changed the management of chronic lymphocytic leukemia (CLL) patients. Despite its clinical efficacy, relapses occur, and outcomes after ibrutinib failure are poor. Although BTK and PLCγ2 mutations have been found to be associated with ibrutinib resistance in a fair percentage of CLL patients, no information on resistance mechanisms is available in patients lacking these mutations. The heat shock protein of 70 kDa (HSP70) and its transcription factor heat shock factor 1 (HSF1) play a role in mediating the survival and progression of CLL, as well as taking part in drug resistance in various cancers. We demonstrated that resveratrol and related phenols were able to induce apoptosis in vitro in leukemic cells from CLL untreated patients by acting on the HSP70/HSF1 axis. The same was achieved in cells recovered from 13 CLL patients failing in vivo ibrutinib treatment. HSP70 and HSF1 levels decreased following in vitro treatment, correlating to apoptosis induction. We suggest an involvement of HSP70/HSF1 axis in controlling resistance to ibrutinib in CLL cells, since their inhibition is effective in inducing in vitro apoptosis in cells from ibrutinib refractory patients. The targeting of HSP70/HSF1 axis could represent a novel rational therapeutic strategy for CLL, also for relapsing patients.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.