Table_1_Efficacy and safety of venetoclax plus hypomethylating agents in relapsed/refractory acute myeloid leukemia: a multicenter real-life experience.docx

Venetoclax (VEN) has been shown to play a synergistic effect in combination with hypomethylating agents (HMAs) in the frontline treatment of acute myeloid leukemia (AML). However, the potential role of this therapy in the relapsed/refractory (R/R) AML setting, still needs to be further unveiled. The aim of the current study was to retrospectively outline the safety profile, response and survival outcomes of R/R AML patients treated with VEN in association with HMAs. Clinical, biological, and molecular data were collected from 57 patients with R/R AML treated with VEN combined with azacitidine or decitabine between 2018 and 2023. The median age of patients was 63 years, 38 (66.7%) received treatment for relapsed disease while 19 (33.3%) for refractory disease, 5 (8.7%) were treated for molecular relapse. A consistent proportion of the cohort was represented by patients with unfavorable prognostic factors such as complex karyotype (36.8%), secondary AML (29.8%), previous exposure to HMAs (38.6%), and relapse after allogeneic stem cell transplant (22.8%). A total of 14 patients achieved CR (24.6%), 3 (5.3%) CRi, 3 (5.3%) MLFS, and 3 (5.3%) PR, accounting for an ORR of 40.4%. The CR/CRi rate was higher in the group treated with azacitidine than in the group treated with decitabine (37.8% vs. 15%). The median OS was 8.2 months, reaching 20.1 months among responding patients. VEN-HMAs treatment allowed to bridge to allogeneic stem cell transplantation 11 (23.9%) of eligible patients, for which a median OS of 19.8 months was shown. On multivariate analysis, ECOG performance status ≥2, complex karyotype and not proceeding to allogeneic stem cell transplantation after therapy with VEN-HMAs were the factors independently associated with shorter OS. Patients treated with the azacitidine rather than the decitabine containing regimen generally displayed a trend toward superior outcomes. The major toxicities were prolonged neutropenia and infections. In conclusion, this study showed how VEN-HMAs could represent an effective salvage therapy in patients with R/R AML, even among some of those patients harboring dismal prognostic features, with a good toxicity profile. Further prospective studies are thus warranted.</p

CK2 expression in MCL and MM and effects of CK2 inhibition on MCL cell survival.

<p>(A-L). Representative immunohistochemistry of CK2α CK2β in normal lymphoid tissue, MCL, MGUS and MM patient specimens. H&E (A), CK2α(B) and CK2β (C) staining of normal reactive lymphoid tissue. Staining for Cyclin D1 (D), CK2α (E) and CK2β (F) in MCL lymph node. Bone marrow biopsy from MGUS stained for H&E (G), CK2α (H) and CK2β (I); MM bone marrow biopsy stained for H&E (J), CK2α (K) and CK2β (L). (H&E and immunoperoxidase stain; original magnification: 10x and 20x; pictures B, C, E, F have an insert at 100x magnification to show details). (M) WB analysis of CK2α expression. From left to right: 1 normal PBMC, 2 normal B lymphocytes (B Lymph), 3 MCL cell lines, and 3 MM cell lines. β-actin was used as a loading control. (N) Quantification of apoptosis through annexin V staining and FACS analysis in CD19⁺ B lymphocytes from five MCL patients treated with the CK2 inhibitor CX-4945 (5 µM). Data represent results of the five separate patients sample (upper panel) and the mean ± SEM of five independent experiments. * indicates p<0.05.</p

Effects of CK2 inhibition on bortezomib-induced proteotoxic/ER stress.

<p>(A, B) Expression of Ire1α, phosphorylated Ser51 eIF2α (p-Ser51 eIF2α), total eIF2α in U-266 (A) and Granta-519 (B) cells treated with the CX-4945 and bortezomib (BZ in the figure) for 6h (A) and 18h (B). (C-E) Expression of poly-ubiquitin and Hsp70 in INA-6 (C), Granta-519 (D) and Rec-1 (E) cells treated with subtoxic and toxic doses of bortezomib for 18h and subsequently incubated with bortezomib and CX-4945 at different concentrations for additional 9 hours. βactin was used as a loading control.</p

Bortezomib-induced NF-κB and STAT3 dependent genes expression is modulated by CK2.

<p>Quantitative Real Time PCR analysis of NF-<b>κ</b>B <i>(IAP-2, TNFα, COX-2, IL-6, Bcl2, NOS-2</i>) and STAT3 (<i>Cyclin D1, IL-6)</i> target gene expression performed on INA-6/HS-5 co-coltures treated with sub-apoptotic doses of bortezomib (BZ in the figure) (2.5 nM, dark bars), CX-4945 5 µM (grey bars) or the combination of the two compounds (grey striped bars) for 18 hours. Data represent mean ± SD of at least 3 independent experiments. * indicates p<0.05. # indicates p<0.05 between samples treated with bortezomib alone and bortezomib together with CX-4945.</p

RNA interference of CK2 in MM INA-6 cells modulates apoptosis and STAT3 phosphorylation.

<p>(A) INA-6 cells were transfected with siGLO Green scrambled or siGLO Green plus CK2α and CK2βsiRNA oligos. 48h after transfection bortezomib 5 nM (BZ) was added to the cultures for 18h. Cells were collected 72h post-transfection. Apoptosis was evaluated through annexin V staining and FACS analysis on siGLO Green positive cells (A) and WB determination of the antiapoptotic markers Mcl1 and Bcl2 (B). In (A) histogram bars represent mean ± SEM of four independent experiments (left panel), while the cytometric histograms show the percentage of annexin V positive cells in a representative experiment (right panel). * indicates p<0.05. ♦ indicates p<0.05 between samples treated with bortezomib 5 nM alone and bortezomib 5 nM together with CK2 α.β siRNA. (B) STAT3 phosphorylation on Ser 727 (p-STAT3 Ser727, total STAT3, CK2α and CK2β levels were determined by WB in INA-6 tranfected with siGLO Green scrambled or siGLO Green plus CK2α and CK2βsiRNA oligos and treated with BZ for 18h. β-actin was used as a loading control.</p

Effects of CK2 and proteasome inhibition on MM and MCL cell proliferation.

<p>Synergistic effect of K27 and bortezomib (BZ in the figure) (A, C, E, G) or CX-4945 and bortezomib (B, D, F, H) on U-266 (A, B), INA-6 (C, D), Jeko-1 (E, F) and Rec-1 (G, H) cell proliferation. Left graphs: dose-response curve of cells incubated for 48 hours with increasing concentrations of K27 or CX-4945 (grey squared curve). Right graphs: dose-response curve of cells incubated for 48 hours with increasing concentrations of bortezomib alone (black squared curve) or K27 or CX-4945 plus bortezomib (black triangle curve). Cell proliferation was assessed by ³H-thymidine-incorporation assay. IC₅₀ for K27 was 6.98 µM and for bortezomib 2.16 nM in U-266; 3.77 µM for K27 and 3.18 nM for bortezomib in INA-6 cells; 4.43 µM for K27 and 4.65 nM for bortezomib in Jeko-1 cells and 3.13 µM for K27 and 11.06 nM in Rec-1 cells. IC50 of CX-4945 was 19.8 µM in U-266, 2.42 µM in INA-6, 2.4 µM in Jeko-1 and 1.46 µM in Rec-1 cells. The CI between K27 and bortezomib was calculated as to be 0.43 for U-266, 0.84 for INA-6, 0.16 for Jeko-1 and 0.5 for Rec-1. The CI between CX-4945 and bortezomib was calculated as to be 0.77 for U-266, 0.05 for INA-6, 0.016 for Jeko-1 and 0.69 for Rec-1.</p

Combined treatment of CK2 inhibitors and bortezomib causes mitochondrial apoptosis of MM and MCL cells.

<p>JC1 staining and FACS analysis of MM cells (INA-6 or INA-6 cocoltured with HS-5 (A) or MCL cell lines (Jeko-1 or Rec-1)(B) treated with the CK2 inhibitors CX-4945 or K27, bortezomib (BZ in the figure) at different concentrations or the combination of CX-4945 or K27 and bortezomib for 18h. Data represent mean ± SEM of at least three independent experiments. * indicates p< 0.05. ♦ indicates p<0.05 between samples treated with bortezomib alone (at the dose indicated in figure) and bortezomib together with CX-4945. # indicates p<0.05 between samples treated with bortezomib 15 nM alone and bortezomib 15 nM together with K27 or CX-4945. • indicates p<0.05 between samples treated with bortezomib 25 nM alone and bortezomib 25 nM together with K27 or CX-4945. (C-D) WB analysis for expression of prosurvival signalling proteins (Bcl2, Mcl1) or pro-apoptotic proteins (Bax, Bak) in MM (C) or MCL (D) cell lines treated with the CK2 inhibitors CX-4945 or K27, bortezomib, or the combination of the two compounds for 18h. GAPDH or β-actin was used as a loading control.</p

Effects of CK2 and proteasome inhibition on MM and MCL cell survival.

<p>Evaluation of apoptosis through WB analysis of PARP cleavage (CL PARP =  Cleaved PARP) (A) in MM cells U-266, INA-6, INA-6 co-cultured with HS-5 (top panels), MCL cells Granta-519, Jeko-1 and Rec-1 (bottom panels); (B, C) annexin V staining and FACS analysis on INA-6 MM cells alone or in co-cultures with HS-5 (B), patient derived plasma cells (C) treated with the CK2 inhibitors CX-4945 (grey bars), the proteasome inhibitor bortezomib (BZ in the figure) at different concentrations (black bars) or the combination of the two compounds (grey striped bars) for 18 hours. In the case of INA-6 grown in co-colture with HS-5, experiments were performed by staining with APC-conjugated anti-CD45 antibody, which is expressed by INA-6 cells but not by stromal cells and with FITC-conjugated annexin V. In C, the apoptotic effect was measured by double staining with CD38 PE and annexin V-FITC. CD38⁺ bright cells were selected. Histogram bars represent mean ± SEM of three independent patient plasma cells (left panel), while the cytometric histograms show the percentage of annexin V positive cells in a representative experiment (right panel); (D) apoptosis of MCL cell lines Granta-519, Jeko-1, Rec-1 treated with the CK2 inhibitors CX-4945 (light grey bars), K27 (dark grey bars), the proteasome inhibitor bortezomib at different concentrations (black bars) or the combination of CX-4945 and bortezomib (grey dotted bars) or K27 and bortezomib (grey striped bars) for 18h. Data represent mean ± SEM of at least three independent experiments. * indicates p<0.05. In B ♦ indicates p<0.05 between samples treated with bortezomib 5 nM alone and bortezomib 5 nM together with CX-4945. In C • indicates p<0.05 between samples treated with bortezomib 2.5 nM alone and bortezomib 2.5 nM together with CX-4945. In D # indicates p<0.05 between samples treated with bortezomib alone and bortezomib together with CX-4945 or K27.</p

Characteristics of MCL cell lines used in the study.

*<p>t(11;14) and rearrangement at 9p22 associated with CCND1 (Cyclin D1) activation and deletion of p15/p16;</p><p>° Cryptic rearrangements of CCND1 and IgH;</p><p>? t(11;14).</p><p>Abbreviations: PB: peripheral blood; MCL: mantle cell lymphoma; LN: lymph node, DLBCL: diffuse large B cell lymphoma; wt: wild-type; del/mut: deleted/mutated.</p

Summary of CK2α, CK2β and STAT3 positivity scores in CD138+ plasma cells of MGUS and Multiple Myeloma cases.

*<p>Nuclear immunolabeling semi quantitatively scored in a four-tier scale: 0 = 0–5%, 1 = 6–33%, 2 = 34–66%, and 3 = 67–100% positive CD138+ cancer cells.</p><p>NA = not assessed.</p