GSK3 inhibitor enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells by overcoming multiple mechanisms of resistance.

In acute myeloid leukemia (AML), the heterogeneity of genetic and epigenetic characteristics makes treatment difficult. The prognosis for AML is therefore poor, and there is an urgent need for new treatments for this condition. Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), targets the CD33 antigen expressed in over 90% of AML cases. GO therefore has the potential to counter the heterogeneity of AML patients. However, a major clinical problem is that drug resistance to GO diminishes its effect over time. Here, we report that the inhibition of glycogen synthase kinase 3 (GSK3) alone overcomes several forms of GO resistance at concentrations without antileukemic effects. The GSK3 inhibitors tested significantly enhanced the cytotoxic effect of GO in AML cell lines. We elucidated four mechanisms of enhancement: (1) increased expression of CD33, the target antigen of GO; (2) activation of a lysosomal function essential for hydrolysis of the GO linker; (3) reduced expression of MDR1 that eliminates calicheamicin, the payload of GO; and (4) reduced expression of the anti-apoptotic factor Bcl-2. A similar combination effect was observed against patient-derived primary AML cells. Combining GO with GSK3 inhibitors may be efficacious in treating heterogeneous AML.

An mTORC1/2 dual inhibitor, AZD2014, acts as a lysosomal function activator and enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells.

Gemtuzumab ozogamicin (GO), an anti-CD33 antibody linked to calicheamicin via an acid-labile linker, is the first antibody-drug conjugate (ADC). The acidic environment inside lysosomes of target cells is an important intracellular determinant of the cytocidal action of GO, as the linker is hydrolyzed under acidic conditions. However, lysosomal activity in acute myeloid leukemia (AML) blasts in GO therapy has been insufficiently evaluated. It has been suggested that lysosome activity is suppressed in AML due to hyperactivation of the phosphoinositide 3-kinase/Akt pathway. We therefore hypothesized that agents which activate lysosomal function would potentiate the cytotoxicity of GO. Here, we found that a clinically useful mTORC1/2 dual inhibitor, AZD2014, reduced pH in the acidic organelles, including lysosomes, as shown by increased LysoTracker fluorescent intensity, and synergistically enhanced the cytotoxic effect of GO in primary leukemia cells. GO-induced cytotoxicity appeared to be enhanced with the increase in lysosomal activity by AZD2014. These results indicate that AZD2014 activated lysosomal function in primary leukemia cells, which in turn enhanced the cytotoxicity of GO. Enhancement of lysosomal activity may represent a new therapeutic strategy in the treatment of GO and other ADCs, particularly in cases with low lysosomal activity.

An mTORC1/2 kinase inhibitor enhances the cytotoxicity of gemtuzumab ozogamicin by activation of lysosomal function.

Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because more than 90% of acute myeloid leukemia (AML) blasts express its target, CD33. Although GO and subsequently developed ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated. In this study, we investigated whether an mTORC1/2 kinase inhibitor, PP242, which was reported to activate lysosomal function, potentiates the cytotoxicity of GO in AML cells. Eight AML cell lines (U937, THP-1, SKM-1, SKK-1, SKNO-1, HL-60, MARIMO and KO52) were treated with GO and PP242. The cytotoxic effect of GO was enhanced by concurrent treatment with a non-cytotoxic concentration (500 nM) of PP242 in most cell lines, except MARIMO and KO52 cells. We then used LysoTracker to label acidic lysosomes in U937, THP-1, SKM-1, MARIMO and KO52 cells. LysoTracker fluorescence was dramatically increased by treatment with PP242 in U937, THP-1 and SKM-1 cells, and the intensified fluorescence was retained with PP242 + GO. In contrast, PP242 did not induce a significant increase in fluorescence in MARIMO cells, consistent with the lack of combinatory cytotoxicity. LysoTracker fluorescence was also increased by PP242 in KO52 cells, which have been reported to strongly express multidrug resistance (MDR). Further, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which in turn triggered cell cycle promotion and cell death. These results indicate that inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle via suppression of GO-induced Chk1 activation. This combination may represent an attractive new therapeutic strategy for the treatment of leukemia.

[Detection of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia by fluorescence in situ hybridization and its clinical significance].

OBJECTIVE: To investigate the presence of p53 gene deletion in Xinjiang patients with chronic lymphocytic leukemia and its clinical significance. METHODS: Interphase fluorescence in situ hybridization (FISH) was used to detect the p53 gene deletion in 77 patients with CLL. Presence of the deletion and its association with clinical and laboratory features as well as prognostic factors were analyzed. Kaplan-Meier method was used to calculate survivals, and the results were compared using a Log-rank test. RESULTS: p53 gene deletion was found in 10 (12.9%) of the patients but none from the control group (P<0.05). The deletion was found in 12.5% (4/32) of ethnic Hans and 13.3% (6/45) of ethnic Uyghurs (P>0.05). No significant different distribution of p53 gene deletion was found in regard to sex, age, ethnicity, peripheral blood cell count (except for Hb) or the levels of lactate dehydrogenase, ß2-micro globulin and CD38 (P>0.05). The deletion rate was higher in the group with high expression of ZAP-70 and patients with advanced stage disease than that in the group of low expression and early-stage CLL (P<0.05). Among 20 patients who received fludarabine therapy, the overall remission rate for those with p53 gene deletion (20%) was lower than those without (75%) (P<0.05). With a median follow-up time of 39.0 (8.0-136.0) months, 11 cases had died (14.3%), among them, 7 cases died from CLL and related complications, and all of them were founded p53 gene deletion. In patients with p53 gene deletion, the progression-free survival (18 months) was shorter than those without the deletion (55 months) (P<0.05). CONCLUSION: The p53 gene deletion has been found in more than 10% of patients with CLL, and the deletion rate did not significantly differ between ethnic Han and Uyghur patients. The deletion is associated with advanced stage of the disease. High-level ZAP-70 expression and the presence of p53 deletion are associated with shorter survival and poor response to fludarabine containing therapy. Therefore, drugs affecting the p53 signaling pathway should be avoided.

[Detection and clinical significance of JAK2 V617F mutation in Chinese and Uyghur patients with chronic myeloproliferative in Xinjiang].

OBJECTIVE: To investigate the frequency of JAK2 V617F gene (hereinafter, the JAK2 gene) mutation in Uyghur patients with chronic myeloproliferative neoplasm (MPN) and its relationship with the clinical characteristics, and further compare differences of mutation rates in Han and Uyghur patients. METHODS: The allele-specific polymerase chain reaction (AS-PCR) was used to detect the JAK2 mutation in 55 cases of Uyghur and 79 cases of Han bcr-abl negative MPN patients. RESULTS: (1) JAK2 mutation rate was 73.1% (98/134); The mutation rates of polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis were 84.3% (43/51), 69.7% (46/66) and 52.9% (9/17), respectively (P < 0.05). (2) The difference of mutation rate in Han \[78.5% (62/79)\] and Uyghur \[65.5% (36/55)\] patients was not significant (P > 0.05). (3) The patients of JAK2 positive have significantly higher count of blood cells, splenomegaly, thrombosis/bleeding and transformation than those of JAK2 negative ones (P < 0.05), but the clinical features between two ethnic groups were not significant (P > 0.05). CONCLUSION: JAK2 gene mutation occurred in the majority of patients with MPN; the mutation rates, clinical features and the complications between Han and Uyghur patients were not significant, which implicated that MPN patients with JAK2 mutation from different regions and ethnics may have the same molecular pathogenesis.