Requirement For CDK6 In MLL-Rearranged Acute Myeloid Leukemia

Abstract

Chromosomal rearrangements involving the H3K4 methyltransferase MLL trigger aberrant gene expression programs in hematopoietic stem and progenitor cells and give rise to an aggressive subtype of acute myeloid leukemia (AML) that is associated with intermediate or poor survival. Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies in the clinic, in part because mutant MLL is difficult to target directly and it is incompletely understood which of the genes affected by altered epigenetic regulation in MLL-rearranged AML are responsible for malignant transformation. To search for essential signaling pathways in MLL-rearranged AML that might serve as new therapeutic targets, we performed loss-of-function RNA interference (RNAi) screens in 5 AML cell lines (NOMO-1, THP-1, OCI-AML3, HL-60, U937) using a lentiviral short hairpin RNA (shRNA) library, and observed that the cell cycle regulator CDK6, but not its functional homolog CDK4, was preferentially required by MLL-AF9pos NOMO-1 and THP-1 cells. The enhanced CDK6 dependence of MLL-rearranged cells was confirmed in an expanded panel of AML cell lines (MLL-rearranged, n=6; MLL wildtype [WT], n=4) that also included cell lines harboring other MLL fusions (MLL-AF4 and MLL-AF6), and the RNAi-induced phenotype was countered by overexpression of an shRNA-resistant CDK6 cDNA. Stable knockdown of MLL-AF9 in MLL-AF9pos cell lines and overexpression of MLL-AF9 in WT MLL-expressing cell lines, normal human CD34pos cells, or Ba/F3 murine pro-B cells led to concordant changes in CDK6 mRNA and protein levels that resembled those of HOXA9, a known MLL-AF9 target, indicating that CDK6 is rendered essential via transcriptional activation by truncated MLL. Analysis of cell cycle distribution, apoptosis induction, and myeloid differentiation demonstrated that the differential growth-inhibitory effect of CDK6 suppression was mainly attributable to myeloid differentiation, as MLL-AF9pos cell lines upregulated CD11b expression and assumed a more mature, macrophage-like morphology upon CDK6 knockdown, effects not observed in WT MLL-expressing cells. Furthermore, the immature phenotype of NOMO-1 cells could be rescued by overexpression of an shRNA-resistant CDK6 cDNA. Consistent with the observations in AML cell lines, knockdown of Cdk6 also impaired the proliferation and in vitro clonogenic activity of primary murine bone marrow (BM) cells stably transduced with MLL-AF9, whereas cells expressing another leukemogenic fusion gene (MOZ-TIF2) and Ba/F3 cells were largely unaffected. We also expressed MLL-AF9 in unfractionated BM derived from Cdk6 knockout mice and observed that colony numbers were gradually reduced in cultures initiated with Cdk6+/- and Cdk6-/- BM compared to WT BM. Furthermore, most of the colonies obtained were small and displayed loose morphology in contrast to the large, dense, blast-like colonies seen in cultures initiated with transduced WT BM. We are currently investigating whether Cdk6 is also required for AML development and propagation in vivo using a murine BM transplantation model of MLL-AF9-induced leukemia. The context-dependent effects of lowering CDK6 expression could be recapitulated in cell lines and primary human AML specimens using palbociclib (also known as PD-0332991), a small-molecule inhibitor of CDK4 and CDK6 enzymatic activity that is in clinical development as an anticancer agent. We are currently devising strategies to combine this compound with cytotoxic chemotherapy as well as other targeted therapeutics, such as small-molecule bromodomain inhibitors, to maximize killing of MLL-rearranged AML cells. Together, our data (1) identify CDK6 as a critical and potentially “actionable” effector of MLL fusion proteins in leukemogenesis, (2) link the catalytic activity of CDK6 to arrested myeloid differentiation in MLL-rearranged AML, and (3) underscore that cell cycle regulators thought to normally act redundantly may have distinct functions in different genetic contexts. Disclosures: No relevant conflicts of interest to declare.

The role of CDK6 in cancer

“…Pharmacological inhibition of CDK6 by the CDK4/6 inhibitor PD‐0332991 (palbociclib), unlocks the blocked differentiation and reduces the leukemic phenotype in human AML cell lines. These findings proposed that CDK6 inhibition provides a clinically applicable therapeutic opportunity for MLL‐rearranged leukemia 38 . A proliferation advantage of leukemic cells through MLL fusion‐driven upregulation of CDK6 has also been shown in MLL‐rearranged infant ALL 39…”

Bortezomib suppresses self‐renewal and leukemogenesis of leukemia stem cell by NF‐ĸB‐dependent inhibition of CDK6 in MLL‐rearranged myeloid leukemia

“…As a serine/threonine kinase, CDK6 is indispensable for the passage of G1 to S phase by phosphorylating retinoblastoma protein 11 . Recently, several studies have revealed that in addition to regulating the cell cycle, CDK6 plays essential roles in apoptosis, 12 reprogramming of cancer cell metabolism, 13 and self‐renewal ability of leukaemia stem cell (LSC) 14 . For example, CDK6 is an indispensable downstream effector in MLL‐rearranged AML, and knockdown of CDK6 reduces the self‐renewal LSC in MLL arrangements‐transformed mouse leukaemia 14 .…”

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation

“…60 Furthermore, CDK6 plays a crucial role in several myeloid diseases, including Jak2 V617F1 myeloproliferative neoplasm, chronic myeloid leukemia, and acute myeloid leukemia, by regulating stem cell quiescence, apoptosis, differentiation, and cytokine secretion. 53,61,62 Using the long-term culture system, it was possible to generate HPC LSKs from the transgenic mouse line Cdk6 2/2 , which represents a powerful tool to analyze specific functions of CDK6 in progenitor cells and allows mechanistic and therapeutic studies tailored specifically to leukemic stem/progenitor cells.…”