Ex vivo characterization of acute myeloid leukemia patients undergoing hypomethylating agents and venetoclax regimen reveals a venetoclax-specific effect on non-suppressive regulatory T cells and bona fide PD-1+TIM3+ exhausted CD8+ T cells.

Acute myeloid leukemia (AML) is an aggressive heterogeneous disease characterized by several alterations of the immune system prompting disease progression and treatment response. The therapies available for AML can affect lymphocyte function, limiting the efficacy of immunotherapy while hindering leukemia-specific immune reactions. Recently, the treatment based on Venetoclax (VEN), a specific B-cell lymphoma 2 (BCL-2) inhibitor, in combination with hypomethylating agents (HMAs) or low-dose cytarabine, has emerged as a promising clinical strategy in AML. To better understand the immunological effect of VEN treatment, we characterized the phenotype and immune checkpoint (IC) receptors' expression on CD4+ and CD8+ T cells from AML patients after the first and second cycle of HMA in combination with VEN. HMA and VEN treatment significantly increased the percentage of naïve CD8+ T cells and TIM-3+ CD4+ and CD8+ T cells and reduced cytokine-secreting non-suppressive T regulatory cells (Tregs). Of note, a comparison between AML patients treated with HMA only and HMA in combination with VEN revealed the specific contribution of VEN in modulating the immune cell repertoire. Indeed, the reduction of cytokine-secreting non-suppressive Tregs, the increased TIM-3 expression on CD8+ T cells, and the reduced co-expression of PD-1 and TIM-3 on both CD4+ and CD8+ T cells are all VEN-specific. Collectively, our study shed light on immune modulation induced by VEN treatment, providing the rationale for a novel therapeutic combination of VEN and IC inhibitors in AML patients.

Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells.

Doxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B-/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B-/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases.

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.

Baseline cluster of differentiation 22 fluorescent intensity correlates with patient outcome after Inotuzumab Ozogamicin treatment.

Antigen-directed target therapy for B-cell acute lymphoblastic leukemia (B-ALL) is now the standard of care for relapsed/refractory (R/R) disease. A comprehensive determination of the target itself is mandatory to aid physician's choice. We determined baseline Cluster of differentiation 22 (CD22) expression percentage and fluorescent intensity on lymphoblasts of 30 patients with R/R B-ALL treated with anti-CD22 immunoconjugate drug Inotuzumab Ozogamicin (INO) and analyzed the impact of both parameters on patient outcome. Most patients (24/30, 80%) had a high leukemic blast CD22-positivity defined as ≥90%. We did not observe a benefit in terms of complete remission, overall survival (OS) and duration of response (DoR) for patients with CD22 ≥ 90% versus CD22 < 90%. Concerning CD22-FI quartile analysis we appreciated a trend for superior response rates in higher quartiles (Q2 -Q4 ) compared to Q1 and a significant benefit in terms of OS and DoR for patients with higher CD22-FI. INO demonstrates to be effective also in patients with lower CD22 expression, but therapeutical benefits are more evident in patients with higher CD22-FI. The evaluation of both CD22 percentage and CD22-FI of the leukemic blast may help physicians in therapeutic choices for R/R B-ALL patients when multiple treatment options are available, although no CD22 expression threshold can currently be identified below which INO should be considered not effective.

INCB84344-201: Ponatinib and steroids in frontline therapy for unfit patients with Ph+ acute lymphoblastic leukemia.

Tyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.

Case Report: A Novel Activating FLT3 Mutation in Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) is among the most common driver genes recurrently mutated in acute myeloid leukemia (AML), accounting for approximately 30% of cases. Activating mutations of the FLT3 receptor include internal tandem duplications (ITD) that map to the auto-inhibitory juxtamembrane (JM) domain or point mutations within the tyrosine kinase domain (TKD). Several FLT3 tyrosine kinase inhibitors have been developed in the last few years, but midostaurin is currently the only one approved for the treatment of newly diagnosed patients harboring FLT3 mutations. Here we describe for the first time a novel in-frame deletion in exon 14 (JM domain) of the FLT3 gene, that we identified in a young woman with CBFb-MYH11-positive AML. We demonstrated that this novel FLT3 variant is pathogenic, since it is responsible for constitutive activation of FLT3 receptor. Finally, ex-vivo studies demonstrated that this novel mutation is sensitive to midostaurin.

Adrenomedullin Expression Characterizes Leukemia Stem Cells and Associates With an Inflammatory Signature in Acute Myeloid Leukemia.

Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.

Novel and Rare Fusion Transcripts Involving Transcription Factors and Tumor Suppressor Genes in Acute Myeloid Leukemia.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795 ∗ ; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Inotuzumab ozogamicin is effective in relapsed/refractory extramedullary B acute lymphoblastic leukemia.

BACKGROUND: Extramedullary involvement of B-cell Acute Lymphoblastic Leukemia (EM-ALL) is a rare occurrence, characterized by dismal outcome and the absence of a defined and shared therapeutic approach. In the landscape of innovative compounds, inotuzumab ozogamicin (IO) is a promising drug, whose mechanism of action relies on the killing of CD22 positive leukemic cells, through the delivery, after cell binding, of a molecule of calicheamicin. CASE PRESENTATION: We report two cases of CD22 positive relapsed EM-ALL treated with IO, obtained as compassionate use. Case 1, a 66 years old woman, affected by Philadelphia (Ph) negative B-ALL, relapsed with extramedullary involvement after 6 standard chemotherapy courses, who reached a complete metabolic response with IO treatment. Case 2, a 67 years old man with Ph positive B-ALL, initially treated with ponatinib, a third generation tyrosine-kinase inhibitor (TKI), obtaining a prolonged deep molecular remission. Nevertheless, for skin relapse during TKI treatment, the patient received local radiotherapy and, shortly after, standard chemotherapy, as multiple abdominal sites of relapse were detected too, with no response. The patient then received IO, obtained as compassionate use, with a good metabolic response. CONCLUSIONS: These two cases suggest a possible key role of IO in the setting of advanced CD22 positive ALL, and underline its potential activity also in patients with EM involvement, relapsed after or refractory to conventional chemotherapy. Despite the well known hepatotoxic effect of the compound (Sinusoid Occlusive Syndrome), neither of them had such adverse event, moreover the second patient safely underwent allogeneic bone marrow transplantation.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia.

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.