Inhibition of Autophagy Does Not Re-Sensitize Acute Myeloid Leukemia Cells Resistant to Cytarabine

Elevated activation of the autophagy pathway is currently thought to be one of the survival mechanisms allowing therapy-resistant cancer cells to escape elimination, including for cytarabine (AraC)-resistant acute myeloid leukemia (AML) patients. Consequently, the use of autophagy inhibitors such as chloroquine (CQ) is being explored for the re-sensitization of AraC-resistant cells. In our study, no difference in the activity of the autophagy pathway was detected when comparing AraC-Res AML cell lines to parental AraC-sensitive AML cell lines. Furthermore, treatment with autophagy inhibitors CQ, 3-Methyladenine (3-MA), and bafilomycin A1 (BafA1) did not re-sensitize AraC-Res AML cell lines to AraC treatment. However, in parental AraC-sensitive AML cells, treatment with AraC did activate autophagy and, correspondingly, combination of AraC with autophagy inhibitors strongly reduced cell viability. Notably, the combination of these drugs also yielded the highest level of cell death in a panel of patient-derived AML samples even though not being additive. Furthermore, there was no difference in the cytotoxic effect of autophagy inhibition during AraC treatment in matched de novo and relapse samples with differential sensitivity to AraC. Thus, inhibition of autophagy may improve AraC efficacy in AML patients, but does not seem warranted for the treatment of AML patients that have relapsed with AraC-resistant disease.

DNA Damage Repair Interference By WEE1 Inhibition with AZD1775 Overcomes Combined Azacitidine and Venetoclax Resistance in Acute Myeloid Leukmeia (AML)

Acute myeloid leukemia (AML) has remained one of the most treatment resistant and deadliest cancers. The survival of AML blast cells is controlled by the balance of anti- and pro-apoptotic proteins. Recently approved Bcl-2 targeted therapy of AML with the Bcl-2 specific inhibitor Venetoclax in combinations has improved patients outcomes. However, a priori and developing resistance to venetoclax combinations with hypomethylating agents (HMA) azacitidine and decitabine challenge this treatment. As such, novel therapies to overcome venetoclax-HMA resistance are urgently needed. We have identified a combination of DNA damage repair interference by WEE1 inhibition with AZD1775, combined with low dose cytarabine (AraC) as an effective strategy to overcome combined venetoclax-azacitidine resistance (VAR). AZD1775 with low dose AraC induced massive apoptosis (by Annexin V and cleaved caspase-3) and almost completely reduced viability and clonogenic growth of primary AML cells. To delineate the molecular mechanism of the synergistic effect of AZD1775/AraC we performed RNAseq analysis of single agent or the combination of AZD1775+AraC in AML cell lines and primary CD34+ selected AML patient cells with the goal to identify deferentially regulated genes indicating a mechanistic underpinning of the potent activity. Only 2 genes were deferentially regulated across cell lines and CD34+ selected cells under AZD1775+AraC treatment: one of these is NR4A1, an orphan nuclear receptor, which we went on to validate as a potential downstream target of Wee1 inhibition. The inactivation of NR4A1 in mice was previously shown to induce AML and to maintain leukemia stem cells. Using qPCR we confirmed that the expression of NR4A1 is upregulated after AZD1775/AraC combo treatment in human leukemic cells. We then demonstrated that activators of NR4A1 (cytosporone B and pPhOCH3) reduce viability of leukemic cells, while NR4A1 inhibitor pPhOH was able to abolish the effect of AZD1775/AraC combo treatment increasing leukemic cell viability]. To investigate the involvement of mitochondria in the effect of AZD1775/AraC treatment we performed the expression of mitochondrial genes and pathway analyses in RNAseq data and found that mitochondrial gene expression, including many genes involved in apoptosis, has most dramatic changes in the combo treatment if compared to the single agents. Subsequently, we have examined the expression of the main BCL-2 family apoptotic genes by qPCR and western blot analysis. We found that AZD1775/AraC induces the expression of Bim isoforms, whereas Bcl-2, Mcl-1 and Bcl-Xl were largely unaffected. NR4A1 was previously shown to translocate to mitochondria, release Bim from Bcl-2 protein binding, as well as convert Bcl-2 to an extreme potent pro-apoptotic form. Finally, we generated several additional VAR cell lines and cells with subclones and demonstrated that AZD1775/AraC combination treatment is able to overcome VAR in almost every clone. Our results show that DNA damage repair interference with Wee1 inhibition has the potential to overcome VAR through a novel mechanisms of AZD1775 increasing NR4A1, freeing pro-apoptotic Bim irrespective of anti-apoptotic Bcl-2 proteins leading to massive apoptotic cell death in AML cells. The precise molecular mechanisms and the involvement of NR4A1 in this phenomenon will be presented at the meeting. Our findings will help to develop new therapeutic strategies in AML treatment and a trial of AZD1775 + AraC in AML is currently ongoing. Disclosures No relevant conflicts of interest to declare.

Structural insights into mutagenicity of anticancer nucleoside analog cytarabine during replication by DNA polymerase η

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.

Acute Myeloid Leukemia Cells Resist Chemotherapy through a Reversible Senescence-like State Maintaining Repopulation Potential

Cancer cells can undergo a senescence-like phenotype in response to genotoxic stress from chemotherapy. Relapse of acute myeloid leukemia (AML) frequently occurs months or years after initial chemotherapy and the functional consequence of chemotherapy-induced senescence (CIS) has not been elucidated. We propose that CIS acts as a stress-survival mechanism in AML, allowing cells to persist in a dormant state with potential to repopulate leukemia post-treatment. To quantify senescence-associated-β-galactosidase (SA-β-gal) activity, we used a fluorogenic substrate (C12-FDG) that enabled sensitive quantification of the senescence marker in viable cells by flow cytometry. We found increased SA-β-Gal activity after exposure to the chemotherapeutic agent cytarabine (AraC) in p53-deficient myeloid leukemia cell lines (K-562 and KG-1), indicating that p53 is not essential for CIS in AML. Using an ex vivo culturing model, we found that patient-derived AML cells demonstrate a greater SA-β-gal response to increasing dosage or duration of AraC treatment until cells undergo apoptosis and exhibit diminished C12-FDG levels. This suggested that CIS is not a distinct condition, but rather a continuous response to the degree of genotoxic stress. ATR kinase activation mediates senescence by enforcing cell cycle arrest in response to genotoxic stress during replication. Treatment of AML specimens with an inhibitor of ATR (VE-821) reduced the initiation of senescence and markedly impaired cell survival after exposure to a moderate dose of AraC. This implied that ATR mediates CIS, independent of p53, to increase stress survival following AraC treatment. In order to characterize the response to genotoxic stress, we treated patient-derived AML cells with AraC, analyzed RNA expression, and performed gene set enrichment analysis (GSEA) with senescence-associated genes compiled from literature. Inflammatory mediators and extracellular matrix proteins are strongly induced after AraC treatment; these are partly related to the senescence-associated secretory phenotype (SASP) and senescence-messaging secretome (SMS) (p<0.001). Thus, primary AML cells also display a senescent gene expression signature after AraC exposure. To model the latent dormant state after chemotherapy, we treated patient-derived AML cells with high doses of AraC (1000 and 10 000 nM for 3 days) that killed the majority of the leukemia population in vitro. Cells surviving initial AraC treatment with 1000 nM persisted in a senescent-like dormant state for 3-4 weeks before initiating leukemia repopulation, while cells treated with 10 000 nM AraC continued to be in a dormant state. To recapitulate the relapse in vivo, we established an AML relapse model using primary human specimens engrafted in NSG mice. Administration of AraC, using a physiologically relevant dose and schedule (60 mg/kg/day x 5 days), to AML-engrafted NSG mice reduced peripheral blood leukemic cells and total body leukemic burden 8 days after initiation of therapy to a small residual leukemia fraction (nadir). This reduction in leukemic burden was reversed after 4 weeks. Gene expression analysis of purified human AML cells at nadir (day 8) were significantly enriched for senescence signatures (p<0.001). This enrichment was reduced by day 29 post-treatment as expression was partially reversed to untreated levels. Notably, GSEA revealed reduction of hematopoietic and leukemia stem cell signatures at nadir, which partially recovered in some AML cases at day 29. Finally, to demonstrate that senescent-like cells maintain leukemia-repopulating potential, we sorted for low (untreated control cells), moderate, and high levels of SA-β-gal activity in C12-FDG+ AML cells after AraC treatment. Transplantation of the sorted cells into NSG mice demonstrated repopulation of leukemia from senescent-like cells. Notably, mice transplanted with equal numbers of high C12-FDG+ senescent-like cells had shorter overall survival compared to mice with moderate or low C12-FDG+ cells. Altogether, our results show that AML cells after chemotherapy can persist in a reversible senescent-like dormant state with leukemia repopulation capacity and reveal a novel mechanism of chemotherapy resistance with therapeutic potential in AML. Disclosures Duy: GlaxoSmithKline: Research Funding. Melnick:Janssen: Research Funding.

The Novel CDK4/6-Inhibitor Abemaciclib Induces Early G1-Arrest in MCL Cell Lines, Sensitizes Cells to Cytarabine Treatment and Is Additive with Ibrutinib

Introduction: Mantle cell lymphoma (MCL) is characterized by t(11;14) resulting in a constitutive cyclin D1 overexpression. The cyclin D1-CDK4/6 complex inactivates Rb through phosphorylation, leading to G1/S-phase transition. Therefore, inhibition of CDK4/6 is an efficient and rational approach to overcome cell cycle dysregulation in MCL. We evaluated the efficiency of the novel CDK4/6 inhibitor abemaciclib in various MCL cell lines and in primary MCL cells in combination with cytarabine (AraC) and ibrutinib. Material & Methods: MCL cell lines (Granta 519, JeKo-1, Maver-1, Mino) and primary MCL cells were exposed to abemaciclib alone and combined with AraC or ibrutinib. Cells were pretreated with abemaciclib and exposed to AraC or ibrutinib with or without consecutive wash-out of the CDK4/6 inhibitor. Proliferation and viability were measured by tryptan blue staining and Cell Titer Glo assay. Flow cytometry was used for cell-cycle (PI-staining) and apoptosis analysis (Annexin V PE/7AAD-staining). Western Blot analysis showed protein expression and phosphorylation status of various downstream proteins. Results: Abemaciclib inhibited cell proliferation by induction of early G1-arrest. Western Blot analysis revealed reduced phosphorylation of Rb on serine 795 without changes in CDK 4 and cyclin D1 expression, in line with reversible cell cycle arrest. IC50-values of sensitive cell lines (JeKo-1, Maver-1, Mino) were <30 nM after 72 h. We observed an almost complete and reversible G1-arrest in all sensitive cell lines by FACS analysis (JeKo-1: G1-phase +51,7 %; S/G2-phase -51,7 % at 31,25 nM after 24 h; G1-phase +35,4 %; S/G2-phase -34,8 % after 72 h), whereas cell viability was not reduced. Wash-out of abemaciclib after 24 h resulted in synchronized S-phase entry in all sensitive cell lines (e.g. Mino: G1-phase -20,4 %; S-phase +30,5 %). The sequential combination of abemaciclib followed by AraC showed strong synergy in Mino cells (CI=0,22 for 31,25 nM abemaciclib and 3,33 µM cytarabine). In contrast, simultaneous exposure to abemaciclib had a protective effect against AraC treatment in all sensitive cell lines, due to an ongoing G1-arrest (Mino: CI=-0,19 for 31,25 nM abemaciclib and 3,33 µM AraC). In primary MCL cells, 31,25 nM of abemaciclib had no impact on cell death. Moreover, no sensitization to AraC was observed as all cells were resting in G0-phase. The combination of abemaciclib induced G1 arrest and ibrutinib had additive or synergistic effects in sensitive cell lines (JeKo-1, Mino and Maver). Conclusion: The novel CDK4/6 inhibitor abemaciclib causes reversible G1 cell cycle arrest without loss of viability at low nanomolar doses. Rationale drug combinations exploiting the sequential effect may achieve major benefits, but drug interactions are complex: Pretreatment with abemaciclib sensitizes MCL cell line cells to AraC whereas simultaneous application protects them from AraC treatment. Further analyses explore the interaction with other targeted approaches (inhibitors of the B-cell receptor pathway) to better understand the underlying molecular mechanisms. Disclosures No relevant conflicts of interest to declare.