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In vivo functional screens to decipher mechanisms of stochastically- and mutationally-induced chemoresistance in Acute Myeloid Leukemia

Periodic Reporting for period 3 - AltCheM (In vivo functional screens to decipher mechanisms of stochastically- and mutationally-induced chemoresistance in Acute Myeloid Leukemia)

Reporting period: 2021-03-01 to 2022-08-31

Acute Myeloid Leukemia (AML) is the most common acute leukemia diagnosed in adults and regroups a heterogeneous group of diseases. This heterogeneity reflects a wide variety of genomic alterations that are responsible for a marked disparity of chemotherapeutic responses and overall cure rate. Thus, a majority of patients relapse after front-line therapy and decease from expansion of a minimal residual disease (MRD) whose mechanisms of emergence and resistance to new rounds of chemotherapy remain poorly understood.

Using innovative functional screening approaches, this project aims at i) exploring the central mechanism - either stochastically- or mutationally-driven - that governs the emergence of MRD, and ii) establishing the cellular mechanisms of MRD persistence despite continuous exposure to chemotherapeutic agents. By addressing such fundamental questions, we will also be able to identify signaling pathways that actively promote chemoresistance in AML, and possibly in other hematopoietic disorders. The comprehensive analysis of their unknown function will potentially define new therapeutic routes in AML, which could work with front-line therapy and targeted therapies to specifically block relapse burden.

To address this fundamental question, our proposal makes use of (i) a stochastic model of chemoresistance in which a fraction of leukemic cells will be the main reservoir of disease relapse regardless of their mutational status [Aim 1] or (ii) a mutational model predetermined to resist by virtue of a collection of chemoprotective mutations which were selected from sequencing studies on human AML samples taken at diagnosis versus relapse [Aim 2].
[Aim 1] The stochastic model of chemoresistance was generated using an AML mouse model driven by a single mutational hit, the MLL-AF9 translocation. In contrast to many other models of AML, MLL-AF9 is by itself sufficient to induce full leukemia transformation. By consecutive rounds of standard AML chemotherapy regimens, an MLL-AF9 leukemic cell population highly resistant in the bone marrow environment was isolated and its transcriptional program was profiled by RNA-sequencing in comparison with naive MLL-AF9-driven leukemic blasts prior to the identification using an in vivo shRNA screen of the set of genes that are functionally required to promote resistance to front-line therapies.

We were able to isolate a set of 42 genes defined as chemosensitizers which were interrogated by computational biology to feature cellular nodes that represent cornerstones of resistant to chemotherapy. We established that most of these genes were engaged in the regulation of RNA splicing and stabilization as well as in the control of ribosome biogenesis and translation. In that regard, we conducted three complementary approaches to fully appreciate the central role of post-transcriptional mechanisms in promoting chemoresistance: i) a qualitative and quantitative bioinformatic analysis of alternative splicing using our RNA-sequencing data, ii) a Ribo-sequencing study intended to characterize mechanisms of translation fidelity and depict the signature of proteins synthesized by translation of mRNA – hereinafter referred to as translatome, and iii) a (phospho)proteomic profiling intended to define altered signaling pathways in chemoresistant cells.

Although the Ribo-sequencing study is still ongoing, the (phospho)proteomic profiling already revealed that the family of mRNA splicing regulator CLK kinases is more expressed and activated in cells resistant to front-line therapies. A gene network analysis combined with an algorithm which estimates the likelihood a substrate to be phosphorylated by a given kinase according to the cellular context then established that three other splicing factors, SRRM1, SRRM2, and PRPF8 which also scored as chemosensitizers in the pooled shRNA screen were all connected within the same functional network to CLK kinases. According to this observation, these hits were individually retested both in naive and resistant cells to establish that they are strict vulnerabilities in resistant cells only. In addition, invalidation of CLK kinases using small-molecule inhibitors or shRNAs altered the viability of both human and murine chemoresistant AML cells and increased markedly their sensitivity to both cytarabine and daunorubicin.

[Aim 2] Modeling a mutationally-determined system of chemoresistance requires that collections of pre-leukemic mutations may cooperate in mediating chemoprotection. Loss-of-function mutations in TET2 gene is among the most frequent pre-leukemic genetic events in patients with diagnosed AML and are also recurrently found in patients with relapse disease. They usually do not emerge as single entities, but are rather combined with other cooperating mutated pre-leukemic hits. This observation supposes that we could determine the combination of pre-leukemic mutations which, when combined with TET2 loss, may functionally confer chemoprotection. Using an innovative in vivo functional screening of a library of pooled mutated Open Reading Frame (ORF) constructs encoding an extensive list of pre-leukemic mutations, we are implementing a novel methodology to nominate combinations of pre-leukemic mutations that can predispose to resistance to front-line therapy. We have generated a conditional Tet2 cKO mouse model which was interrogated using the pooled library of 28 mutated ORFs flanked with an individual barcode and encoding the mutant proteins referenced as preleukemic mutations. After treatment with chemotherapy, these mice were euthanized and the relative proportion of the pre-leukemic mutations that are expected to promote chemoresistance in combination with the invalidation of Tet2 will be determined by sequencing. These pending sequencing results will allow us to nominate an extensive panel of mutated hit candidates whose biological functions in promoting resistance to front-line therapies will be dissected during the second grant period.
[Aim 1] The next and final lines of investigation will consist in:

(i) Depicting comprehensively the translatome of chemoresistant and naive cells and its molecular intricacy with our (phospho)proteomic data.

(ii) Defining whether the suppression of our candidate genes is sufficient to perturb the translational landscape of chemoresistant cells, thereby bringing into light their central function as regulators of AML cell resistance to front-line therapies through the control of the post-transcriptional machinery.

(iii) Analyzing the effect of CLK kinase inhibition in primary xenografts from four paired primary and relapse AML patient samples injected in immunodeficient mice. Positive results from this preclinical experiment could legitimate the launching of a phase-1/2 clinical trial evaluating the safety and efficacy of a CLK inhibitor, SM08502, in combination with the standard front-line therapies in patients with AML (partnership between Samumed, LLC. and the AML department and the Clinical Investigation Center of Saint-Louis Hospital (CLIP2 INCa).

[Aim 2] The next lines of investigation will consist in:

(i) Validating the sequencing results and isolating the set of pre-leukemic mutations conferring resistance to front-line therapies.

(ii) Making use of these chemoresistant clones to decipher the cellular mechanisms involved in this mode of chemoresistance by deploying transcriptomic and shRNA-based functional analyses.
Overview of the Study