Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Duplication of 8q24 in Chronic Lymphocytic Leukemia: Cytogenetic and Molecular Biologic Analysis of MYC Aberrations.

Chronic lymphocytic leukemia (CLL) with cytogenetics findings, such as complex karyotype and deletions of TP53 or ATM, is associated with adverse clinical outcomes. Additional chromosomal abnormalities further stratify patients into groups with diverse prognoses. Gain of 8q24 is one of the abnormalities considered as prognostically unfavorable. In our study, we performed a FISH analysis in an initial cohort of 303 consecutive CLL patients and determined the frequency of +8q to be 6.3 %. Our analysis confirmed the association with TP53/ATM aberrations and CK, as the frequency of +8q reached 26.7 % in an extended delTP53/ATM+CK cohort. M-FISH analysis enabled the identification of partner chromosomes where the segment of the duplicated 8q arm was localized. More detailed mapping of the gained 8q region using the M-BAND method determined the smallest amplified region 8q23-8qter. We observed significantly shorter overall survival (OS; 9.0 years in +8q-positive vs. 10.6 years in +8q-negative; p=0.02) and detected slightly higher MYC mRNA/protein levels in +8q-positive vs. +8q-negative patients.

Synthesis and Profiling of Highly Selective Inhibitors of Methyltransferase DOT1L Based on Carbocyclic C-Nucleosides.

Histone methyltransferase DOT1L is an attractive therapeutic target for the treatment of hematological malignancies. Here, we report the design, synthesis, and profiling of new DOT1L inhibitors based on nonroutine carbocyclic C-nucleoside scaffolds. The experimentally observed SAR was found to be nontrivial as seemingly minor changes of individual substituents resulted in significant changes in the affinity to DOT1L. Molecular modeling suggested that these trends could be related to significant conformational changes of the protein upon interaction with the inhibitors. The compounds 22 and (-)-53 (MU1656), carbocyclic C-nucleoside analogues of the natural nucleoside derivative EPZ004777, and the clinical candidate EPZ5676 (pinometostat) potently and selectively inhibit DOT1L in vitro as well as in the cell. The most potent compound MU1656 was found to be more metabolically stable and significantly less toxic in vivo than pinometostat itself.

ATR-CHK1 pathway as a therapeutic target for acute and chronic leukemias.

Progress in cancer therapy changed the outcome of many patients and moved therapy from chemotherapy agents to targeted drugs. Targeted drugs already changed the clinical practice in treatment of leukemias, such as imatinib (BCR/ABL inhibitor) in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), ibrutinib (Bruton's tyrosine kinase inhibitor) in chronic lymphocytic leukemia (CLL), venetoclax (BCL2 inhibitor) in CLL and acute myeloid leukemia (AML) or midostaurin (FLT3 inhibitor) in AML. In this review, we focused on DNA damage response (DDR) inhibition, specifically on inhibition of ATR-CHK1 pathway. Cancer cells harbor often defects in different DDR pathways, which render them vulnerable to DDR inhibition. Some DDR inhibitors showed interesting single-agent activity even in the absence of cytotoxic drug especially in cancers with underlying defects in DDR or DNA replication. Almost no mutations were found in ATR and CHEK1 genes in leukemia patients. Together with the fact that ATR-CHK1 pathway is essential for cell development and survival of leukemia cells, it represents a promising therapeutic target for treatment of leukemia. ATR-CHK1 inhibition showed excellent results in preclinical testing in acute and chronic leukemias. However, results in clinical trials are so far insufficient. Therefore, the ongoing and future clinical trials will decide on the success of ATR/CHK1 inhibitors in clinical practice of leukemia treatment.

Novel CHK1 inhibitor MU380 exhibits significant single-agent activity in TP53-mutated chronic lymphocytic leukemia cells.

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.