Kinase Suppressor of Ras 2 promotes self-renewal and clonogenicity of small-cell lung carcinoma

Small-cell lung carcinoma (SCLC) tumors are heterogeneous, with a subpopulation of cells primed for tumor initiation. Here, we show that Kinase Suppressor of Ras 2 (KSR2) promotes the self-renewal and clonogenicity of SCLC cells. KSR2 is a molecular scaffold that promotes Raf/MEK/ERK signaling. KSR2 is preferentially expressed in the ASCL1 subtype of SCLC (SCLC-A) tumors and is expressed in pulmonary neuroendocrine cells, one of the identified cells of origin for SCLC-A tumors. The expression of KSR2 in SCLC and pulmonary neuroendocrine cells (PNECs) was previously unrecognized and serves as a novel model for understanding the role of KSR2-dependent signaling in normal and malignant tissues. Disruption of KSR2 in SCLC-A cell lines inhibits the colony forming ability of tumor propagating cells (TPCs) in vitro and their tumor initiating capacity in vivo. The effect of KSR2 depletion on self-renewal and clonogenicity is dependent on the interaction of KSR2 with ERK. These data indicate that the expression of KSR2 is an essential driver of SCLC-A tumor propagating cell function, and therefore may play a role in SCLC tumor initiation. These findings shed light on a novel effector promoting initiation of ASCL1-subtype SCLC tumors, and a potential subtype-specific therapeutic target. Impact Statement Manipulation of the molecular scaffold KSR2 in ASCL1-subtype small-cell lung cancer cells reveals its contribution to self-renewal, clonogenicity, and tumor initiation via ERK signaling.


Introduction
Small-cell lung carcinoma (SCLC) affects current and former heavy smokers, accounting for 13% of all lung cancers 1 .There have been few improvements in SCLC detection, treatment, and survival in almost 40 years, leading to its classification as a recalcitrant cancer in 2012 1 .The five-year relative survival rates for SCLC patients with localized, regional, and distant disease are 30%, 18%, and 3%, respectively (American Cancer Society, 2024).Currently, SCLC patients are treated with first line therapy (cisplatin or carboplatin combined with etoposide chemotherapy, plus anti-PDL1 antibody, atezolizumab), second (topotecan), and third line (PD1 antagonist, nivolumab) therapies, and for some patients thoracic radiation therapy 2 .Although SCLC tumors are responsive to therapy initially, residual disease quickly develops resistance leading to the low five-year survival 3 .Substantial efforts have been made to characterize SCLC tumors and identify targets that may be selectively toxic to tumor cells while preserving normal lung tissue [4][5][6][7][8] .Rigorous and innovative basic science using state-of-the-art genetically-engineered mouse (GEM) models, and an extensive set of cell lines have led to key discoveries regarding the cells-of-origin and the common recurring mutations that underlie SCLC 7,[9][10][11][12][13][14][15][16][17][18] .These discoveries have yielded comprehensive genomic profiles and a durable classification of SCLC subtypes based on the differential expression of four key transcription factors, ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y) 7,13,17,18 .
Lineage tracing and single cell RNA sequencing (scRNA-seq) in genetically modified mice showed that a rare pulmonary neuroendocrine cell (PNEC) subpopulation, NE stem cells, actively responds to lung injury of the epithelia by expanding, migrating, and undergoing Notch-dependent transit amplification to regenerate the damaged epithelium 19 .This effort additionally identified NE stem as a cell-of-origin for SCLC following Trp53, Rb1 and Notch mutations causing constitutive activation of stem cell renewal and altered deprogramming 19 .SCLC tumors in some GEM models have a small population of tumor propagating cells (TPCs) essential to the initiation, long-term propagation, and metastatic capacity of the tumor, while the bulk non-TPC population is highly proliferative but incapable of establishing tumors in vivo 20 .TPCs have been implicated in the initiation and growth of SCLC as well as therapy resistance [21][22][23][24][25][26][27] .Tumor propagating cells are also implicated in epithelial-to-mesenchymal transition (EMT) and metastasis 27 .Their slower cycling and self-renewing ability enhances DNA repair, rendering these cells resistant to DNA damage-dependent chemo and radiation therapy 23,24,27,28 .Thus, SCLC TPCs offer a unique population within which to search for new targets, which in combination with current standard-of-care therapies may yield a durable and effective strategy for therapy.reduced 3-to 12-fold from 1/10-1/39 in NTC to 1/112-1/140 in KSR2 KO cells (Fig. 5C, Supplementary Fig. 1C,  D).These data indicate that KSR2 is a critical effector of tumor initiating capacity in human SCLC tumor propagating cells.KSR2-ERK interaction is necessary for SCLC clonogenicity.KSR2 knockdown reduces activation of ERK (Fig. 6A).With serum starvation, control and KSR2 knockdown samples are reduced to a basal level of ERK activation (Fig. 6A).ERK can be stimulated with addition of serum in control cells, while KSR2 knockdown cells activate ERK poorly after serum addition (Fig. 6A).Treatment with calcium ionophore ionomycin also induces ERK activation in control cells, but KSR2 knockdown inhibits this response (Fig. 6A).Cells expressing KSR2r rescue ERK activation in each condition (Fig. 6B).To assess the importance of the interaction between KSR2 and ERK, a DEF domain mutant (570 FIFP/AAAP 573) 29 KSR2 construct deficient in its ability to bind ERK (FIF570) was further mutated to be resistant to binding hairpin sh5 (FIF570r) and expressed in KP1 sh5 cells.Cells expressing FIF570r are unable to rescue ERK activation when endogenous KSR2 is depleted (Fig. 6C).Disruption of KSR2/ERK interaction prevents ERK activation in response to serum stimulus or treatment with ionomycin.Immunoprecipitation of the FLAG epitope tag on this construct and the full length KSR2 demonstrated reduced 6hosphor-ERK associated with the ERK-binding mutant construct (FIF570r) (Fig. 6D).Colony formation was significantly reduced in the FIF570r cells after dox-induced targeting of endogenous KSR2, suggesting that KSR2 interaction with ERK is necessary for the clonogenic capacity of SCLC TPCs (Fig. 6E).Disruption of KSR2 significantly reduced the number of colonies formed by TPCs (Fig. 6F).KSR2r rescued colony formation (Fig. 6F), while FIF570r was unable to restore colony formation to KP1 cells (Fig. 6F).These data indicate that KSR2-dependent ERK signaling is an important contributor to colony formation by TPCs.

Discussion
Kinase Suppressor of Ras proteins KSR1 and KSR2 have unique and overlapping functions.Both function as scaffolds for Raf/MEK/ERK signaling 29,30 promoting phosphorylation of ERK.KSR2 is expressed in the brain, pituitary gland, and adrenal gland, and some neuroendocrine cells and tissues 31,32,44 .Here we show that disruption of KSR2 in SCLC-A reduces TPC clonogenicity and self-renewal in vitro, and tumor initiation in vivo.Further work demonstrates that the clonogenic capacity of TPCs depends upon the interaction of KSR2 with ERK.Activating Ras mutations are rare in SCLC tumors, therefore Raf/MEK/ERK signaling is likely activated by extracellular stimuli, suggesting that a molecular scaffold, such as KSR2, is required to amplify this signaling pathway 45 .The role of ERK signaling in SCLC is incompletely understood, but is implicated in cell proliferation, differentiation, survival, and drug resistance 46 .Our data reveal KSR2-dependent ERK signaling is involved in self-renewal and clonogenicity of the SCLC-A TPC population.This observation contrasts with previous studies that tested the efficacy of targeting Raf/MEK/ERK signaling in SCLC and concluded that ERK activation can be both pro-and anti-proliferative toward SCLC 45 .Treatment with an ERK inhibitor was unable to induce apoptosis in human SCLC-A cell lines H209 and H69 47 .In contrast, our data are consistent with observations that SCLC-A cell line proliferation was significantly reduced in vitro and in in vivo tumor xenografts by ARHGEF19 disruption and downstream reduced Raf/MEK/ERK signaling 48 .Activation of Raf/MEK/ERK by endoplasmic reticulum (ER) stress has also been reported to promote SCLC cell survival 49 .Raf/MEK/ERK signaling may play an essential role in promoting metastasis of SCLC tumors 45 .CXCL12 induces ERK activation in SCLC cells, which correlates with increased invasion through extracellular matrix 50 .Increased Raf/MEK/ERK signaling induced by expression of an activated Raf construct has been reported to cause growth arrest and reduce expression of neuroendocrine markers in SCLC 51,52 .These contrasting observations may suggest that the intensity, frequency, localization, or duration of ERK activation and its effects may be context dependent.Further, our findings suggest that Raf/MEK/ERK signaling may be selectively important for the TPC population, while dispensable for the bulk tumor population.
The effect of KSR2 depletion on ERK activation is surprising as SCLC-A cells express both KSR2 and KSR1.We do not detect KSR1 compensation for KSR2 loss, suggesting that KSR2 is required for TPC function in SCLC-A cells.Our data suggest that either KSR2 is contributing to TPC function by promoting signaling that KSR1 cannot influence, or that KSR2 and KSR1 coordinate function to conduct efficient signaling.KSR1 and KSR2 are capable of heterodimerization with Raf, resulting in a conformational change of KSR proteins that allows phosphorylation of MEK [53][54][55] .The dimerization of KSR proteins with Raf orients the Raf protein so that its catalytic site is not in close proximity to the phosphorylation site on MEK to complete the phosphorylation, which necessitates MEK phosphorylation by Raf through a trans interaction 53 .KSR2-BRAF heterodimerization results in an increase of MEK phosphorylation via the KSR2-mediated relay of signal from BRAF to release the activation segment of MEK for phosphorylation 53 .KSR2 can also homodimerize via the same interface that interacts with BRAF, however this creates a different quaternary structure when interacting with MEK and it's unknown how this may affect the availability of MEK for phosphorylation 53 .BRAF homodimers, or KSR1-BRAF heterodimers, should generate a quaternary structure sufficient for availability of MEK for phosphorylation by an additional BRAF protein 53 .Via the same interaction interface, it is possible that KSR1 and KSR2 heterodimerize.Our incomplete understanding of how KSR2 homodimers or KSR1-KSR2 heterodimers may impact MEK phosphorylation makes it difficult to predict the effect of KSR2 loss on MEK phosphorylation and downstream ERK activation.However, KSR2 depletion has a deleterious effect on ERK activation in SCLC-A cells.It is notable that we fail to detect KSR2 expression in the NEUROD1-subtype of SCLC (SCLC-N), while they retain expression of KSR1 (Fig. 1E, F).This observation suggests that SCLC-N cells are capable of compensating for loss of KSR2 with KSR1 expression, or that transition to the NEUROD1-subtype SCLC results in upregulation of other compensatory signaling pathways that negate the dependency on KSR2 for ERK signaling.
ASCL1 and NEUROD1 drive distinct transcriptional profiles in SCLC 14,56,57 .SCLC-A cells can be converted to SCLC-N cells by elevation of MYC 57 .MYC amplification has been associated with poor patient outcome, therapy resistance, and tumor progression [58][59][60] .KSR2 expression seems to be lost during transition from SCLC-A to SCLC-N.The initiation of switching from SCLC-A subtype to SCLC-N subtype is incompletely understood, although recent studies implicate that epigenetic mechanisms are involved 61 .The expression of KSR2 in SCLC-A is thought to be driven by its defining transcription factor ASCL1, as KSR2 is a transcriptional target of ASCL1 33,34 .ASCL1 and KSR2 are also expressed in SCLC-A cell-of-origin, PNECs.PNECs respond to injury of the lung epithelia by expanding, migrating, and undergoing Notch-dependent transit amplification to regenerate the damaged epithelium 19 .Although KSR2 plays a role in SCLC TPC function, its role in normal PNECs is undetermined.KSR2 knockout mice have metabolic defects 30,31 but have no reports of abnormal lung pathology.Studies determining the effect of KSR2 manipulation on lung repair are required to determine if KSR2 plays a role in the function of PNECs in response to damage.
If KSR2 is dispensable for normal lung repair, it represents a potential therapeutic target for SCLC-A tumors.The discovery that cell surface proteins CD24, CD44, and EpCAM can be used to enrich and isolate SCLC-A TPCs significantly advanced investigation of potential therapeutic vulnerabilities within this population 19 .Cisplatin treatment does not enrich TPCs indicating that TPCs are not a reservoir for drug tolerant persister cells 19 , however TPCs are capable of repopulating tumors after treatment suggesting that they are not preferentially sensitive to the chemotherapeutic.Isolation of TPCs allows for interrogation of potential therapies that may be selectively detrimental to the TPC population.The distinct transcriptional profiles of the SCLC subtypes are therapeutically relevant, as there are a number of MYC-associated vulnerabilities in ASCL1 low tumors which differ from vulnerabilities identified in ASCL1 high tumors 62 .Subtype specific vulnerabilities have been reported for all four SCLC subtypes 63 suggesting that further investigation into subtype specific therapeutic options, and their clinical relevance, is necessary.The preferential expression of KSR2 in SCLC-A, and the role it plays in TPC function provide rationale for further interrogating KSR2 as a SCLC-A specific therapeutic target.
JQ1, an inhibitor of bromodomain and extra-terminal (BET) proteins, reduced the frequency of TPCs in vivo and suppressed the ability of single TPCs to form colonies in vitro 19 .Our data demonstrate that KSR2 disruption reduces frequency of TPCs and reduces colony formation in vitro and tumor initiation in vivo.The effect of KSR2 on TPC clonogenicity is dependent on interaction with ERK, revealing a role for ERK activation in TPC function.These results suggest that targeting of KSR2 or its effector ERK may be TPC-specific therapeutic strategies and may create a more durable therapeutic response when used in conjunction with standard of care chemotherapies.The data provides a rationale for interrogating the effects of targeting KSR2 in genetically engineered mouse models of SCLC-A 12,14,64 .KSR2 disruption may work well in combination with cisplatin to target TPCs, reducing tumor burden and recurrence.

Cell culture
Murine small-cell lung carcinoma cell lines KP1 and KP3 were a gift from J. Sage (Stanford University).Human small-cell lung carcinoma cell lines H209 and H1963 were a gift of John Minna (UT Southwestern).The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and grown at 37°C with ambient O 2 and 5 % CO 2 .Serum starved cells were grown in above conditions, with serum removed for 16 hours prior to cell lysis.Serum shocked cells were grown normally followed by boosting FBS concentration to 50% for 15 minutes prior to cell lysis.Ionomycin (ThermoFisher Scientific I24222) treatment was given at a 1 uM dose for 15 minutes prior to cell lysis.All cells were routinely tested for mycoplasma.No further authentication of cell lines was performed by the authors.

Generation of KSR2 shRNA knockdown and CRISPR/Cas9 knockout cell lines
Individual SMARTvector human inducible lentiviral shRNAs suitable for targeting murine and human KSR2 expressed in piSMART hEF1a/TurboGFP vector were stably transfected into KP1, KP3, and H209 SCLC cell lines with PEI.Cells were selected for expression of the shRNAs using 0.25 µg/mL of puromycin.48 hours after doxycycline induction, cells were selected again by flow cytometry sorting for GFP+ cells.Knockdown of KSR2 was confirmed by western blot.KSR2 cDNA (MSCV KSR2 IRES YFP) and ERK binding deficient KSR2 cDNA (MSCV KSR2 FIF570 IRES YFP) was made resistant to binding of hairpin sh5 by introducing three point mutations in the binding region.Point mutations were introduced using the QuikChange Lightning Site Directed Mutagenesis Kit (Agilent #210518) according to the manufacturer's protocol.Sequencing of the construct confirmed the correct point mutations were made with no additional mutations.MSCV KSR2 IRES YFP resistant to binding hairpin sh5 (KSR2r) and MSCV KSR2 FIF570 IRES YFP resistant to binding hairpin sh5 (FIF570r) were transfected into HEK-293T cells using trans-lentiviral packing system (ThermoFisher Scientific).The virus was collected 48 hours post transfection and used to infect KP1 sh5 cells with 8 µg/mL Polybrene for 72 hours.KP1 sh5 cells expressing the KSR2r or FIF570r construct were selected for using flow cytometry sorting YFP+ cells.Presence of the KSR2r or FIF570r expression after doxycycline induced downregulation on endogenous KSR2 was confirmed via western blotting.To generate knockout cells, sgRNA sequences targeting KSR2 or non-targeting control (NTC) were inserted into lentiCRISPR v2 (Addgene #52961).The constructs were PEI transfected into HEK293T cells along with psPAX2 lentiviral packaging construct (Addgene #12259) and pMD2.G envelope construct (Addgene #12259).Lentivirus-containing media was harvested at 72-h and used to infect the H209 cells with 8 µg/mL Polybrene.Cells were selected for expression of the sgRNAs using 2 ug/mL of puromycin, and western blot was used to confirm KSR2 knockout.

Analysis of KSR2 expression in normal tissues and mouse tumors
GTEx portal was used to display the relative expression of human KSR2 mRNA (TPM) in brain-cortex and lung tissue.In a murine model, neuroendocrine specific reporter, Chga-GFP was used to identify PNECs.GFP+ neuroendocrine cells from 3 mouse lungs were isolated by flow cytometry.qPCR was performed to measure mRNA expression of Ksr2, Cgrp, Chga, Syp, and Spc in GFP+ neuroendocrine cells and GFP-lung epithelial cells.RPR2, RPM and RPMA mouse tumor data is derived from Ireland et al., Cancer Cell, 2020 37 and Olsen et al., Genes Dev, 2021 38 and deposited in NCI GEO: GSE149180 and GSE155692.

SCLC sequencing analysis
RNA sequencing data from human primary tumor samples 13 was analyzed for KSR2 expression based on high and low ASCL1 expression or high and low MYC expression.RNA sequencing data of human SCLC cell lines (DepMap Portal, Broad Institute MIT) was segregated by SCLC subtype and analyzed for KSR2 mRNA levels.
Fluorescence Activated Cell Sorting SCLC cell lines were incubated 20 minutes on ice in PBS with DAPI (3uM), PE-CD24 (1:400) PE-Cy7-CD44 (1:300) and APC-EpCAM (1:100).Cells were resuspended in PBS and flow cytometry of SCLC cell lines was performed using a 100 µm nozzle on a BD FACSAria II using FACSDiva software.Debris were excluded by gating on forward scatter area versus side scatter area.Doublets were excluded by gating on forward scatter area versus side scatter height.Viable cells were identified by exclusion of DAPI stained cells.CD24 high CD44 low cells were included by sequential gating followed by EpCAM high TPCs.Compensation was performed using single stain and fluorescence-one (FMO) controls.Positive gates were set based on the negative unstained sample.Data were analyzed using FlowJo software.

Colony Formation Assay
For colony formation assays, SCLC cells were dissociated by gentle pipetting.Live TPCs were sorted using a 100 μm nozzle on a BD FACSAria II.TPCs were sorted individually into 96 well plates filled with regular media (200 μl/well) containing DMSO or doxycycline (DOX) (1 µg/mL).50 µL fresh media with or without DOX was added to the wells every 10 days.Three weeks later, colony numbers were assessed using CellTiter-Glo 2.0 reagent (Promega #G9242) and luminescence was measured (POLARstar Optima plate reader) according to the manufacturer's protocol.CellTiter-Glo® readings greater than 300 Relative Luminescence Units (RLUs) in colony forming assays were considered colonies.

Extreme Limiting Dilution Analysis In vivo
The viable cell number was assessed by replicate cell counts on a hemocytometer using Trypan Blue exclusion.Viable cell number was used to derive a titration of cell numbers for implantation.Cells were diluted in 50 μl media (RPMI +10% FBS) and 50 μl Cultrex PathClear BME (Trevigen # 3632-005-02).Six eight-weekold NCG mice were injected subcutaneously into the shoulders and flanks.3 replicates for each dilution were used.Mice were provided drinking water with 5% sucrose, or sucrose plus doxycycline (2 mg/kg).Injection sites were palpated biweekly to monitor for tumor growth and all mice were sacrificed when any one tumor reached 1 cm.Tumors that formed were scored as 1 and the absence of tumor formation was scored as 0 for the extreme limiting dilution analysis (ELDA).Tumors that formed were analyzed for expression of KSR2 by western blot and tumors in the doxycycline treated group which did not maintain knockdown were scored as 0. ELDA software was used to estimate TPC frequency for control and doxycycline treated groups.