Natural-Based Indirubins Display Potent Cytotoxicity toward Wild-Type and T315I-Resistant Leukemia Cell Lines

Abstract Abstract 3500 Hsp90, one of the best-characterized molecular chaperones, plays indispensable roles in folding and assembly, intracellular transport, stabilization, and degradation of proteins, and therefore, facilitating cell signaling. Hsp90 is also involved in tumorigenesis by stabilizing oncogenic client proteins. Thus, Hsp90 inhibition has been considered a promising therapeutic strategy for different types of cancer including leukemia. PU-H71 is a novel HSP90 inhibitor with high specificity for oncogenic Hsp90. We investigated the effect of PU-H71 in acute myelogenous leukemia (AML), in particularly, AML stem cells (AML-SCs) that are known to give rise to AML blasts, are refractory to conventional therapies, and thus likely to account for AML relapses. The effect of PU-H71 was evaluated using a panel of 12 leukemia cell lines. Among the 12 leukemia cell lines tested, MOLM-13 and MV4-11 cells were the most sensitive (LD50s 253 nM and 120 nM respectively). Both MV4-11 and MOLM-13 carry FLT3-ITD mutation (occurring in ∼40% AML cases) and MLL translocations (occurring in ∼20% AML cases). Both MLL and FLT3 have been reported as client proteins of Hsp90. However, other leukemia cell lines with MLL rearrangements such as THP-1 and a MLL-ENL cell line derived from MLL-ENL transformed human CD34+ cord blood cells exhibited resistance to PU-H71 treatment (LD50 > 2 μM). The data suggested that FLT3-ITD+ AML samples may display higher sensitivity to Hsp90 inhibition. To confirm the higher sensitivity of FLT3-ITD+ AML cells to Hsp90 targeted therapy, 15 primary AML patient samples (8 FLT3-ITD mutants and 7 wild type FLT3) were treated with increasing concentrations of PU-H71. Cell viability on different cell populations was evaluated using multiparameter flow cytometry at 48 hours after treatment with PU-H71. The average LD50 of PU-H71 in FLT3-ITD+ AML cells was 492 nM (95% CI, 127.636 – 856.364). In contrast, the average LD50 in AML samples with wild type FLT3 was 2.795 μM (95% CI, 1.058 – 4.532). The near 6-fold difference between LD50s for PU-H71 was significant (p=0.0068). Importantly PU-H71 also killed FLT3-ITD+ AML stem and progenitor cells more effectively. Furthermore, PU-H71 treatment decreased the ability to form colonies in FLT3-ITD+ AML specimens more effectively than FLT3 WT AMLs (97.6% and 79.3% decrease relative to control respectively; N=3; P=0.0236). Importantly, PU-H71 had minor toxicity to normal blood mononuclear cells and normal cord blood hematopoietic stem cells. FLT3-ITD+ cell lines and primary AML cells treated with 0.5 μM PU-H71 showed a substantial decrease of phosphorylated forms of Erk1/2, JNK, AKT, p70RSK, NF-κB(p65) and Stat5, which was observed within 4 hours post PU-H71 treatment, whereas the phosphorylation levels of MAPK p38 remained unaffected. Immunoblotting and phosphoflow assays corroborated the inhibition of AKT and Stat5 signaling by PU-H71 in stem and progenitor populations. In summary, FLT3-ITD+ AML cells display a stronger response to PU-H71, suggesting that the FLT3-ITD mutation results in a higher dependency on Hsp90 to stabilize the aberrant signaling elicited by constitutive activation of FLT3. Our data suggests that PU-H71 represents a novel therapy for FLT3-ITD+ AML patients with the potential to ablate AML-SCs. Disclosures: Roboz: EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

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The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.3523.3523 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3523-3523

Author(s):

Danielle Garshott ◽

Nicole Melong ◽

Tania T. Sarker ◽

Yue Xi ◽

Amy Brownell ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemia Cells ◽

Wild Type ◽

Leukemia Cell Lines ◽

Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

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Inhibition of growth of human leukemia cell lines by retrovirally expressed wild-type p16INK4A

Leukemia ◽

10.1038/sj.leu.2400840 ◽

1997 ◽

Vol 11 (10) ◽

pp. 1673-1680 ◽

Cited By ~ 19

Author(s):

AF Gombart ◽

R Yang ◽

MJ Campbell ◽

JD Berman ◽

HP Koeffler

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Human Leukemia ◽

Wild Type ◽

Human Leukemia Cell ◽

Inhibition Of Growth ◽

Leukemia Cell Lines

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DOCK2 Interacts with FLT3 and Modulates the Survival of FLT3-Expressing Leukemia Cells

Blood ◽

10.1182/blood.v124.21.3584.3584 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3584-3584

Author(s):

Min Wu ◽

Max Hamaker ◽

Li Li ◽

Donald Small ◽

Amy S. Duffield

Keyword(s):

Cell Lines ◽

Conflicts Of Interest ◽

Receptor Gene ◽

Leukemia Cell ◽

Leukemia Cells ◽

Wild Type ◽

Knock Down ◽

Leukemia Cell Lines ◽

Significant Difference ◽

Flt3 Expression

Abstract The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying FLT3/ITD mutations have a poor prognosis. Despite continuing progress in the development of more effective FLT3 inhibitors, long-term success in inhibition of FLT3 activity in AML patients is still elusive. In order to achieve a better understanding of FLT3 biology and more effective strategies for the inhibition of FLT3 activity, a screen was performed on leukemia cell lines to search for FLT3-interacting proteins. One of the proteins identified in the screen was dedicator of cytokinesis 2 (DOCK2). The DOCK family of proteins acts as guanine nucleotide exchange factors (GEFs) for Rho family of GTPases, which includes Rac GTPases. DOCK2 expression is limited to hematopoietic cells, and is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells, and cell-cell adhesion and bone marrow homing of various immune cells. We first verified that DOCK2 is expressed in primary AML samples from patients, and co-immunoprecipitation experiments showed that DOCK2 interacts with both wild-type FLT3 and FTL3/ITD in these cells. Co-immunoprecipitation experiments using leukemia cell lines demonstrated that DOCK2 interacts with FLT3, FLT3/ITD, FLT3/D835Y and FLT3/D835H, and that it predominantly interacts with the unphosphorylated form of FLT3. Knock-down of DOCK2 by shRNA did not significantly affect the growth of cell lines that lack expression of FLT3, but greatly reduced growth of cell lines expressing amplified wild type FLT3 (Sem K2), FLT3/D835H (HB11;19) and FLT3/ITD (MV4;11). Accordingly, colony formation assays revealed that cell lines with elevated expression of wild type or mutant FLT3 produced fewer, smaller and more compact colonies when the expression of DOCK2 was decreased, while colonies from cell lines lacking FLT3 expression showed no significant difference in response to the knock-down of DOCK2. Furthermore, an Annexin V binding assay indicated that reduction in DOCK2 expression level greatly sensitized cells with elevated FLT3 activity (MV4;11 and Sem K2) to cytarabine, resulting in increased apoptosis, but no significant sensitization was observed in cell lines that lack FLT3 expression. These findings demonstrate that DOCK2 interacts with FLT3 in leukemia cell lines, and suggest that this interaction has important roles in regulating the survival of leukemia cells with elevated FLT3 activity, both alone and in combination with conventional anti-leukemic agents. Therefore, DOCK2 is a potential therapeutic target for AML treatment, and better understanding of the interaction between DOCK2 and FLT3 may contribute to the development of novel strategies to effectively inhibit FLT3 activity in AML patients. Disclosures No relevant conflicts of interest to declare.

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Frequent mutations in the p53 gene in human myeloid leukemia cell lines

Blood ◽

10.1182/blood.v79.9.2378.2378 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2378-2383 ◽

Cited By ~ 123

Author(s):

K Sugimoto ◽

H Toyoshima ◽

R Sakai ◽

K Miyagawa ◽

K Hagiwara ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Direct Sequencing ◽

Leukemia Cell ◽

P53 Gene ◽

Wild Type ◽

Rt Pcr ◽

Leukemia Cell Lines ◽

Myeloid Leukemia Cell ◽

P53 Mrna

Abstract The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor- dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.

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A new strategy to resume the apoptosis activity of p53 in leukemia cell lines retaining wild-type p53

Leukemia Research ◽

10.1016/j.leukres.2006.09.014 ◽

2007 ◽

Vol 31 (8) ◽

pp. 1156-1158 ◽

Cited By ~ 5

Author(s):

Ze-Jun Liu ◽

Hai-Ming Xin ◽

Jie Chen ◽

Xin Lu ◽

Shan Zhong ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Wild Type ◽

Leukemia Cell Lines ◽

New Strategy ◽

Wild Type P53

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Frequent mutations in the p53 gene in human myeloid leukemia cell lines

Blood ◽

10.1182/blood.v79.9.2378.bloodjournal7992378 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2378-2383 ◽

Cited By ~ 4

Author(s):

K Sugimoto ◽

H Toyoshima ◽

R Sakai ◽

K Miyagawa ◽

K Hagiwara ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Direct Sequencing ◽

Leukemia Cell ◽

P53 Gene ◽

Wild Type ◽

Rt Pcr ◽

Leukemia Cell Lines ◽

Myeloid Leukemia Cell ◽

P53 Mrna

The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor- dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.

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Apoptosis Induction in Human Lymphoma and Leukemia Cell Lines by Transfection via Dendrosomes Carrying Wild-Type p53 cDNA

Biotechnology Letters ◽

10.1007/s10529-005-4689-y ◽

2006 ◽

Vol 28 (1) ◽

pp. 61-66 ◽

Cited By ~ 1

Author(s):

Mohammad Massumi ◽

Abed Ali Ziaee ◽

Mohammad Nabi Sarbolouki

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Apoptosis Induction ◽

Wild Type ◽

Leukemia Cell Lines ◽

Human Lymphoma ◽

Wild Type P53

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Development of an MDM2 Degrader for Treatment of Acute Leukemias

Blood ◽

10.1182/blood-2021-147522 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1866-1866

Author(s):

Bridget Marcellino ◽

Xiaobao Yang ◽

He Chen ◽

Karie Chen ◽

Claudia Brady ◽

...

Keyword(s):

Cell Lines ◽

Ubiquitin Ligase ◽

Research Funding ◽

Leukemia Cell ◽

Data Safety Monitoring Board ◽

Feedback Mechanism ◽

Dependent Manner ◽

Wild Type ◽

Leukemia Cell Lines ◽

Direct Binding

Abstract Introduction Acute myeloid leukemias (AMLs) are characterized by suppressed cell death pathways which promote leukemic blast survival. TP53 acts as a tumor suppressor gene in AML and is found mutated or deleted in 10-15% of patients. In a majority of cases though, TP53 is wild-type. Other mechanisms including MDM2 over-expression lead to reduced TP53 activity. MDM2 acts as a negative regulator by direct binding of TP53 and mediating TP53 degradation through ubiquitination. MDM2 itself is a transcriptional target of TP53 as a negative feedback mechanism limiting the function of TP53. Small molecule inhibition of MDM2 , blocking its ability to bind TP53, can activate TP53 and trigger cell cycle arrest and apoptosis through increased transcription of TP53 target genes. Increased MDM2 expression has been observed in hematologic malignancies including AML, providing rationale for clinical trials with MDM2 inhibitors. These agents such as RG7388 and AMG232 have shown efficacy as monotherapy and in combination. However, these agents have also exhibited toxicity and have yet to demonstrate sufficient benefit for their approval. To create more effective agents against MDM2, we have developed an MDM2 degrader XY-27 that functions as a proteolysis-targeting chimera (PROTAC). Based on relatively higher expression in AML compared to other cancer types, we selected VHL as the E3 ubiquitin ligase target for XY-27 , as this may improve specificity and potency in AML. Results The PROTAC degrader XY-27 concurrently binds MDM2 and VHL, and by bringing these targets in proximity, VHL can then ubiquitinate MDM2, leading to its degradation by the proteasome. XY-27 can mediate degradation of MDM2 in a concentration dependent manner in the U937 leukemia cell line (Fig 1a). MDM2 degradation with XY-27 is blocked by proteasome inhibition and competitive binding of the VHL ligand. A control compound, which only differs in that it cannot bind to VHL, lacks degrader activity. Although MDM2 is itself an E3 ligase, VHL expression is not appreciably changed with XY-27 (Fig 1a). Treatment with XY-27 leads to apoptosis and decreased proliferation of leukemia cell lines in a TP53 dependent manner. Inhibition of MDM2 leads to up-regulation of TP53 and in TP53 wild-type cells, downstream targets CDKN1A (p21) and PUMA. MDM2 is also up-regulated through a feedback mechanism. XY-27 demonstrated greater potency than the MDM2-binding inhibitor AMG232 in the MOLM13 and MV4-11 leukemia cell lines (Fig 1b). Treatment with XY-27 led to higher levels of TP53 and p21 protein than with AMG232. CRISPR-mediated knock-out of VHL leads to reduced XY-27 potency. XY-27 also shows efficacy when combined with other chemotherapeutic agents such as azacytidine and cytarabine. In a long-term co-culture model with an OP9 feeder layer, XY-27 was capable of inducing apoptosis in primary patient AML samples (Fig 1c). Conclusion We describe a new MDM2 PROTAC, XY-27 that demonstrates TP53 dependent activity against leukemia cells. It also demonstrates increase potency compared to an MDM2 binding inhibitor. Utilization of the PROTAC system has potential advantages through selection of the VHL E3 ubiquitin ligase. Because of negative feedback mechanisms involving TP53 and MDM2, direct binding inhibitors of MDM2 may be limited in activity through continued accumulation of MDM2. PROTAC degraders have catalytic activity and may overcome this inhibition by continued degradation of the target MDM2, and thus achieve greater TP53 activity. Figure 1. Activity of the MDM2-PROTAC XY-27 in leukemia. (a) Western blot from treatment of U937 leukemia cells with XY-27 for 24 hrs, at various concentrations (5 nM to 1 μM), resulting in the degradation of MDM2. (b) Dose response curves from treatment of MOLM13 and MV4-11 cell lines with XY-27 (blue) and AMG232 (red) for 48 hrs, demonstrating greater potency of XY-27. (c) Induction of apoptosis in primary AML cells treated with XY-27 at 1μM using a co-culture system for 3 days. *p<.05 Figure 1 Figure 1. Disclosures Hoffman: Protagonist Therapeutics, Inc.: Consultancy; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding.

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